R2 Protein Research Articles

Antibodies by Non-Febrile, Smear-Negative Individuals from a Malaria Epidemic-Prone Setting in Ethiopia are Strongly Reactive to Plasmodium falciparum Blood-Stage Vaccine Candidate Antigens

Background: Plasmodium falciparum malaria remains a major public health concern globally though there is some decline due to scale-up of control efforts. Evaluation of the anti-malarial immune profile, in populations residing in epidemic-prone areas in the dry season or at the time when vector control largely reduced man-mosquito contact, would help predict malaria burden when future epidemics occur. Methods: A cross-sectional study was designed to investigate antibody responses to four P. falciparum blood stage vaccine candidate antigens in non-febrile individuals from Shewa Robit in north-central Ethiopia where malaria transmission was expected at a minimal level as a result of the sampling season and effective vector control. Blood samples were analyzed microscopically for Plasmodium detection. The enzyme-linked immunosorbent assay (ELISA) was used to measure immunoglobulin (IgG) antibodies to apical membrane antigen 1 (AMA1), glutamaterich protein (GLURP) R2 region and merozoite surface protein 2 (MSP2) allelic variants. Results: Study participants were smear-negative for Plasmodium infection. While 51 (22%) of the participants reported that they had never been exposed to clinical malaria in life, 177 (78%) reported at least one clinical malaria episode with laboratory confirmed P. falciparum infection. The antigens tested were well-recognized by the test sera although significant differences were observed in antibody prevalence and level between the different antigens and there was inter-individual variability. IgG response to the antigens showed age-related pattern but without evidence of relation with status and frequency of reported past exposure to clinical malaria. Conclusion: The data suggests that individuals in an epidemic-prone malaria setting have reactive and stable antibodies that readily recognize P. falciparum blood-stage vaccine candidate antigens in the absence of slidepositivity. Analysis of age-related pattern in antibody level showed positive association with age but unrelated with increasing frequency of reported episode suggesting the role of intrinsic age-related factors in malaria immune maturation.

Bioinformatics Analysis of UL39 Gene from Duck Plague Virus

The structures and characteristics of the large subunit of ribonucleotide reductase (R1), encoded by UL39 gene from duck plague virus (DPV) were analyzed and predicted by using a series of free bioinformatics software packages and bioinformatics tools. The DPV UL39 gene has a base composition of 681 adenine (27.99%), 503 cytosine (20.67%), 593 guanine (24.37%) and 656 thymine (26.96%). The online analysis of the physico-chemical properties demonstrates that the protein has 40 potential phosphorylation sites and 4 N-glycosylation sites when the threshold of prediction score is above 0.5; without the signal peptide and the transmembrance region. The phylogenetic tree proved that DPV R1 protein had a close evolutionary relationship with the Mardivirus genus of the Alphaherpesviruses. In conclusion, all those results will provide some valuable information for the further research of UL39 gene.

Effects of PKC on TNF-α stimulated IP3 R1 protein expression in human mesangial cells

Objective To explore the effects of TNF-α on the expression of IP3 R1 mRNA and protein in human mesangial cells (HMCs) and elucidate the role of protein kinase C (PKC) in this signal pathway.Methods Quantitative real-time polymerase chain reaction and immunoblot assay were used to examine the effects of TNF-α on IP3R1 mRNA and protein expression.Depletion PKC,the selective inhibitor of PKCα Safingol and inhibitor of PKCδ Rottlerin,overexpression of dominant negative mutant of PKC to examine the mechanism of signal transduction of TNF-α-regulated IP3 R1 in HMCs.PKCα activation was assayed by Western blot.Results TNF-α increased IP3R1 mRNA and protein expression in HMCs,effects that were blocked by prolonged incubted chronic PMA,Safingol and also by domain negative PKCα construct.TNF-α promoted PKCα activation with maximal PKCα phosphorylation that occurred 8 h post-stimulation.Conclusion TNF-α increased the expression of IP3 R1 and this was mediated through the PKCα activation signaling pathways in HMCs. Key words: Hepatorenal syndrome; TNF-α; Human mesangial cells; IP3 R1; PKCα

Endothelial cell–selective adhesion molecule in diabetic nephropathy

Endothelial cell-selective adhesion molecule (ESAM) contributes to the integrity of tight junctions and modulates endothelial function. ESAM has been linked to experimental diabetic nephropathy; its soluble fraction is related to atherosclerosis in humans. In this cross-sectional observational study, we describe for the first time serum ESAM in type 2 diabetic patients with different stages of chronic kidney disease (CKD) and its relationship to vascular endothelial growth factor-A (VEGF-A). Materials and methods We included diabetic patients with different stages of CKD and controls. History, laboratory evaluation, serum ESAM and VEGF-A and urinary albumin/creatinine ratio were obtained. Endothelial cell-selective adhesion molecule was higher in non-CKD diabetic patients 13.80 (6.15-18.70) ng/mL (n=45) than controls 7.30 (4.60-9.40) ng/mL (n=48), P=0.001. VEGF-A had a similar pattern: 71.3 (54.75-120.70) vs. 43.20 (30.1-65.90) pg/mL, P<0.0001. ESAM was 10.4 (5.6-17.4) ng/mL in predialysis CKD patients (n=59) and 22.35 (8.55-29.95) ng/mL in dialysis patients (n=36), P<0.001. Patients with glomerular filtration rate (GFR)<15 mL/min had the highest ESAM (P=0.003). ESAM was similar in normoalbuminuric, microalbuminuric and proteinuric patients. ESAM was directly correlated with the duration of diabetes (r(2)=0.048, P=0.009), C-reactive protein (r(2)=0.028, P=0.05), VEGF-A (r(2)=0.040, P=0.01) and inversely with HbA1C (r(2)=0.036, P=0.03), haemoglobin (r(2)=0.062, P=0.005) and albumin (r(2)=0.0·40, P=0.026). In multiple regression diabetes duration, HbA1C and VEGF-A were significant predictors of ESAM. In controls, ESAM was inversely related to VEGF (r(2)=037, P=0.01). Endothelial cell-selective adhesion molecule and VEGF-A are higher in patients with diabetes than in controls. The highest ESAM is found in dialysis patients. ESAM correlates with diabetes duration and control, inflammation and VEGF-A in patients with diabetes, but not in controls.

A Dynamic C‐Terminal Segment in the Mycobacterium tuberculosis Mn/Fe R2lox Protein Can Adopt a Helical Structure with Possible Functional Consequences

Mycobacterium tuberculosis R2-like ligand-binding oxidase (MtR2lox) belongs to a recently discovered group of proteins that are homologous to the ribonucleotide reductase R2 proteins. MtR2lox carries a heterodinuclear Mn/Fe cofactor and, unlike R2 proteins, a large ligand-binding cavity. A unique tyrosine-valine cross link is also found in the vicinity of the active site. To date, all known structures of R2 and R2lox proteins show a disordered C-terminal segment. Here, we present two new crystal forms of MtR2lox, revealing an ordered helical C-terminal. The ability of alternating between an ordered and disordered state agrees well with bioinformatic analysis of the protein sequence. Interestingly, ordering of the C-terminal helix shields a large positively charged patch on the protein surface, potentially used for interaction with other cellular components. We hypothesize that the dynamic C-terminal segment may be involved in control of protein function in vivo.

Oxysterols modulate calcium signalling in the A7r5 aortic smooth muscle cell-line

Prolonged exposure to oxidized low density lipoprotein (oxLDL) can alter various aspects of cell biology, including modification of vasomotor responses and downregulation of calcium channel proteins in aortic smooth muscle cells. However, the components of oxLDL responsible for these effects have not been fully elucidated. The study reported here aimed at examining the consequences of extended exposure to oxysterols, cholesterol oxidation products whose levels are elevated in oxLDL as compared to unmodified LDL, on calcium signalling mechanisms in A7r5 cells, a model aortic smooth muscle cell-line. Within 24h of exposure, all three oxysterol congeners tested caused an elevation in the resting cytoplasmic Ca(2+) concentration. These oxysterols also inhibited Ca(2+) transients in response to arginine vasopressin and bradykinin, and some but not all congeners ablated Ca(2+) signals triggered by platelet activating factor, the ryanodine receptor calcium channel agonist 4-choloro-meta-cresol, or thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+) uptake. The effects of long-term exposure to the oxysterol congener 7β-hydroxycholesterol on arginine vasopressin stimulated Ca(2+) signals were mainly at the level of Ca(2+) release from intracellular stores rather than on Ca(2+) influx mechanisms. Of the calcium signalling proteins tested, only the type 1 ryanodine receptor and the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) were significantly downregulated by 24h exposure to oxysterols. Decreases in IP(3)R1 protein triggered by 7β-hydroxycholesterol were both time and concentration dependent, occurring over a concentration range encountered within atherosclerotic lesions. IP(3)R1 downregulation by certain oxysterols is mediated by proteasomal proteolysis, since it can be abolished by co-incubation with epoxomicin. Overall, these data demonstrate that major oxysterol components of oxLDL cause long-term alterations in Ca(2+) signalling in a model aortic smooth muscle cell. Such effects could contribute to the pathology of atherosclerotic disease.

Studies of Ribonucleotide Reductase in Crucian Carp—An Oxygen Dependent Enzyme in an Anoxia Tolerant Vertebrate

The enzyme ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides, the precursors for DNA. RNR requires a thiyl radical to activate the substrate. In RNR of eukaryotes (class Ia RNR), this radical originates from a tyrosyl radical formed in reaction with oxygen (O2) and a ferrous di-iron center in RNR. The crucian carp (Carassius carassius) is one of very few vertebrates that can tolerate several months completely without oxygen (anoxia), a trait that enables this fish to survive under the ice in small ponds that become anoxic during the winter. Previous studies have found indications of cell division in this fish after 7 days of anoxia. This appears nearly impossible, as DNA synthesis requires the production of new deoxyribonucleotides and therefore active RNR. We have here characterized RNR in crucian carp, to search for adaptations to anoxia. We report the full-length sequences of two paralogs of each of the RNR subunits (R1i, R1ii, R2i, R2ii, p53R2i and p53R2ii), obtained by cloning and sequencing. The mRNA levels of these subunits were measured with quantitative PCR and were generally well maintained in hypoxia and anoxia in heart and brain. We also report maintained or increased mRNA levels of the cell division markers proliferating cell nuclear antigen (PCNA), brain derived neurotrophic factor (BDNF) and Ki67 in anoxic hearts and brains. Electron paramagnetic resonance (EPR) measurements on in vitro expressed crucian carp R2 and p53R2 proteins gave spectra similar to mammalian RNRs, including previously unpublished human and mouse p53R2 EPR spectra. However, the radicals in crucian carp RNR small subunits, especially in the p53R2ii subunit, were very stable at 0°C. A long half-life of the tyrosyl radical during wintertime anoxia could allow for continued cell division in crucian carp.

Host Protein BSL1 Associates with Phytophthora infestans RXLR Effector AVR2 and the Solanum demissum Immune Receptor R2 to Mediate Disease Resistance

Plant pathogens secrete effector proteins to modulate plant immunity and promote host colonization. Plant nucleotide binding leucine-rich repeat (NB-LRR) immunoreceptors recognize specific pathogen effectors directly or indirectly. Little is known about how NB-LRR proteins recognize effectors of filamentous plant pathogens, such as Phytophthora infestans. AVR2 belongs to a family of 13 sequence-divergent P. infestans RXLR effectors that are differentially recognized by members of the R2 NB-LRR family in Solanum demissum. We report that the putative plant phosphatase BSU-LIKE PROTEIN1 (BSL1) is required for R2-mediated perception of AVR2 and resistance to P. infestans. AVR2 associates with BSL1 and mediates the interaction of BSL1 with R2 in planta, possibly through the formation of a ternary complex. Strains of P. infestans that are virulent on R2 potatoes express an unrecognized form, Avr2-like (referred to as A2l). A2L can still interact with BSL1 but does not promote the association of BSL1 with R2. Our findings show that recognition of the P. infestans AVR2 effector by the NB-LRR protein R2 requires the putative phosphatase BSL1. This reveals that, similar to effectors of phytopathogenic bacteria, recognition of filamentous pathogen effectors can be mediated via a host protein that interacts with both the effector and the NB-LRR immunoreceptor.

Mammalian ribonucleotide reductase subunit p53R2 is required for mitochondrial DNA replication and DNA repair in quiescent cells

In postmitotic mammalian cells, protein p53R2 substitutes for protein R2 as a subunit of ribonucleotide reductase. In human patients with mutations in RRM2B, the gene for p53R2, mitochondrial (mt) DNA synthesis is defective, and skeletal muscle presents severe mtDNA depletion. Skin fibroblasts isolated from a patient with a lethal homozygous missense mutation of p53R2 grow normally in culture with an unchanged complement of mtDNA. During active growth, the four dNTP pools do not differ in size from normal controls, whereas during quiescence, the dCTP and dGTP pools decrease to 50% of the control. We investigate the ability of these mutated fibroblasts to synthesize mtDNA and repair DNA after exposure to UV irradiation. Ethidium bromide depleted both mutant and normal cells of mtDNA. On withdrawal of the drug, mtDNA recovered equally well in cycling mutant and control cells, whereas during quiescence, the mutant fibroblasts remained deficient. Addition of deoxynucleosides to the medium increased intracellular dNTP pools and normalized mtDNA synthesis. Quiescent mutant fibroblasts were also deficient in the repair of UV-induced DNA damage, as indicated by delayed recovery of dsDNA analyzed by fluorometric analysis of DNA unwinding and the more extensive and prolonged phosphorylation of histone H2AX after irradiation. Supplementation by deoxynucleosides improved DNA repair. Our results show that in nontransformed cells only during quiescence, protein p53R2 is required for maintenance of mtDNA and for optimal DNA repair after UV damage.

Correction: Spectroscopic Studies of the Iron and Manganese Reconstituted Tyrosyl Radical in Bacillus Cereus Ribonucleotide Reductase R2 Protein

Correction: Spectroscopic Studies of the Iron and Manganese Reconstituted Tyrosyl Radical in Bacillus Cereus Ribonucleotide Reductase R2 Protein

Evaluation of ERCC1 and RRM1 as prognostic biomarkers in urothelial cancer (UC) treated with adjuvant platinum and gemcitabine chemotherapy.

e15005 Background: Adjuvant (adj) treatment of UC generally entails platinum (plat)- and gemcitabine (gem)-based chemotherapy (chemo). Two candidate prognostic and predictive biomarkers are the excision repair cross-complementation group 1 gene (E1) and the regulatory subunit of ribonucleotide reductase (R1). Studies in advanced UC patients (pts) suggest that high E1 and R1 expression confers chemoresistance to cisplatin (cis) and gem respectively. We identified UC pts treated with adj chemo to assess for differences in time to recurrence (TTR) and overall survival (OS) based on tumoral E1/R1 expression and chemotherapy regimen. Methods: 184 pts, who received adj plat or gem for T2-4NxM0 UC from 1984-2009, were identified. Tissue microarrays were created from their primary bladder tumor and involved nodal tissue. E1 and R1 protein expression was assessed using AQUA (HistoRx). The median (med) values were established a priori as the cutoff for high and low expression. Medical records were retrospectively reviewed for pathology, treatment, and outcomes data. Results: 154 pts who had both clinical and gene expression data were eligible for analysis. Pts received cis (134), carboplatin (carbo, 20) and gem (62) based regimens. Med age was 65 yrs. The majority were male (79%) and had pT3 (48.7%) or nodal disease (53.2%). At med followup of 3.9 yrs, 53.4% have recurred. Med TTR and OS were 1.2 and 2.1 yrs. Primary tumor E1/R1 Spearman correlation was moderate at 0.49 but less between primary and nodal disease (E1 0.34, R1 0.23). Irrespective of chemo, E1 and R1 expression were not prognostic for TTR (E1 p=0.8, R1 p=0.3) or OS (E1 p=0.25, R1 p=0.83). Degree of E1 expression did not impact TTR or OS in plat-treated pts nor did R1 expression in gem-treated pts. Stage predicted increased TTR and OS (&lt;0.00001). Cis-treated pts had significantly longer TTR (p=0.02) than carbo-treated pts. Conclusions: Tumoral E1 and R1 expression were not prognostic biomarkers for UC pts treated with adj plat and gem chemotherapy in our cohort. Our data was limited by missing tissue in 22% of planned pts. Reproducibility by a second laboratory and heterogeneity analysis between primary and nodal tissue will be presented.

Arterial Stiffness Is Associated With Increased Monocyte Expression of Adiponectin Receptor mRNA and Protein in Patients With Coronary Artery Disease

Arterial stiffness and carotid intima-media thickness (IMT) constitute validated cardiovascular prognostic markers. Adiponectin and its receptors 1 (AdipoR1) and 2 (AdipoR2) are involved in coronary artery disease (CAD). We investigated whether AdipoR1 and R2 mRNA and protein expression are associated with arterial stiffness, IMT and extent of coronary atherosclerosis. We studied 71 patients (61 men, 10 women) with angiographically proven CAD. We measured: (i) monocyte expression of AdipoR1 and AdipoR2 mRNA (quantitative real-time PCR) and protein expression (flow cytometry) (iii) adiponectin, metalloproteinase-9 (MMP-9) and C-reactive protein (CRP) blood levels, (iv) carotid-femoral artery pulse wave velocity (PWV) and carotid IMT. Patients with multi-vessel CAD had higher AdipoR1 and AdipoR2 mRNA than those with single-vessel (P < 0.05). PWV was associated with AdipoR1 mRNA (r = 0.474), AdipoR1 protein (r = 0.228), AdipoR2 mRNA (r = 0.716), AdipoR2-protein (r = 0.261), adiponectin (r = 0.236), and MMP-9 (r = 0.350) (P < 0.05, for all correlations). After adjustment for age, sex, waist-hip ratio, and mean blood pressure both AdipoR1 and AdipoR2 mRNA remained independent determinants of PWV (R(2) = 0.35 and R(2) = 0.57, P < 0.05). IMT was also associated with AdipoR2 mRNA, AdipoR2 protein, and MMP-9 (P < 0.05). Increased expression of ADR2 mRNA significantly related to MMP-9 (r = 0.210), and CRP (r = 0.531) (P < 0.05). Increased mRNA and protein expression of adiponectin receptors is related with increased aortic stiffness, coronary and peripheral atherosclerosis in patients with CAD. The interrelation of AdipoR2 with inflammatory markers, PWV and IMT suggests a compensatory increase of these receptors to counteract the excess inflammatory and atherogenic process in CAD. Thus, adiponectin receptors may provide a potential therapeutic target of agents activating their beneficial action.American Journal of Hypertension 2012; doi:10.1038/ajh.2012.42.

Dietary fat and sugar induce obesity and impair glucose tolerance in prepubertal pigs

A translational swine model of childhood obesity was used to determine the interaction between dietary energy source, body adiposity, and oral glucose tolerance (OGT). Prepubertal female pigs weaned at 21 d of age were fed control (C), refined sugar (S), fat (F), and sugar‐fat (HE) diets in a 2×2 factorial arrangement for 16 wk. Calories from fat were 8.9% for C, 5.6% for S, 35.5% for F and 32.3% for HE. Calories from sugar were 36.0% for S and 30.7% for HE. Adding fat or sugar increased (P&lt;0.003) calorie intake indicating impaired ability to regulate energy consumption. Daily fat accretion and percent body fat were higher (P&lt;0.03) in all treatments compared to C, and in HE compared to S. Ultrasound backfat depth was associated (P&lt;0.001) positively with percent body fat (r2 = 0.909) and negatively with percent body protein (r2 = −0.912). Fat and sugar intake raised (P&lt;0.02) plasma cholesterol levels. Glucose area under the curve (AUC) in response to OGT at 14 wk was higher (P&lt;0.03) in F (+15%) and HE (+32%) pigs compared to C. Glucose AUC from S‐fed pigs was not different (P=0.2) from C‐ or F‐fed pigs. Adding sugar, fat, or their combination to diets increased (P&lt;0.008) glucose AUC. Inclusion of fat in the diet reduced (P&lt;0.001) insulin AUC during OGT, while sugar had no effect. Inclusion of fat and refined sugar in pig diets increases body adiposity and impairs glucose homeostasis. College of Agriculture and Life Sciences

Spectroscopic Studies of the Iron and Manganese Reconstituted Tyrosyl Radical in Bacillus Cereus Ribonucleotide Reductase R2 Protein

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. We studied the R2F subunit of B. cereus reconstituted with iron or alternatively with manganese ions, then subsequently reacted with molecular oxygen to generate two tyrosyl-radicals. The two similar X-band EPR spectra did not change significantly over 4 to 50 K. From the 285 GHz EPR spectrum of the iron form, a g 1-value of 2.0090 for the tyrosyl radical was extracted. This g 1-value is similar to that observed in class Ia E. coli R2 and class Ib R2Fs with iron-oxygen cluster, suggesting the absence of hydrogen bond to the phenoxyl group. This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν7a = 1500 cm−1) was found to be insensitive to deuterium-oxide exchange. Additionally, the 18O-sensitive Fe-O-Fe symmetric stretching (483 cm−1) of the metallo-cofactor was also insensitive to deuterium-oxide exchange indicating no hydrogen bonding to the di-iron-oxygen cluster, and thus, different from mouse R2 with a hydrogen bonded cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g 1-value of ∼2.0094. The tyrosyl radical microwave power saturation behavior of the iron-oxygen cluster form was as observed in class Ia R2, with diamagnetic di-ferric cluster ground state, while the properties of the manganese reconstituted form indicated a magnetic ground state of the manganese-cluster. The recent activity measurements (Crona et al., (2011) J Biol Chem 286: 33053–33060) indicates that both the manganese and iron reconstituted RNR R2F could be functional. The manganese form might be very important, as it has 8 times higher activity.

Application of synchronous fluorescence spectroscopy for the determination of some chemical parameters in PDO French blue cheeses

The present study was aimed at investigating the potential of using synchronous fluorescence spectroscopy (SFS) coupled with multivariate statistical analyses for the determination of some chemical parameters (pH, fat, dry matter, protein and soluble nitrogen) of French blue-veined cheeses belonging to four brands (FA-Fourme d’Ambert, FM-Fourme de Montbrison, BA-Bleu d’Auvergne and BC-Bleu des Causses). Three partial least square regression models with leave-one-out cross-validation technique were considered in the present study. The first one including the “Fourme cheeses” (FA and FM), the second one including the “Blue cheeses” (BA and BC) and the last one including the 4 Blue-veined cheeses (FA, FM, BA and BC). The models qualities were investigated principally by the R2 (coefficient of determination) and the RPD (ratio of standard deviation to root-mean-square error of cross-validation) factors. The results showed that SFS succeeded to predict ash and protein in Blue (ash: R2 = 0.90, RPD = 3.17; protein R2 = 0.80, RPD = 2.24) or Fourme cheeses (ash: R2 = 0.81, RPD = 2.29; protein R2 = 0.81, RPD = 2.26) when considered individually, while SFS failed to predict all the physicochemical parameters when the two groups were analyzed jointly.

R2 and R2/R1 hybrid non-autonomous retrotransposons derived by internal deletions of full-length elements

BackgroundR2 is a non-long terminal repeat (non-LTR) retrotransposable element that inserts site specifically into the 28S genes of the ribosomal (r)RNA gene loci. Encoded at the 5' end is a ribozyme that generates the precise 5' end by self-cleavage of a 28S gene cotranscript. Sequences at the 3' end are necessary for the R2 protein to bind RNA and initiate the target primed reverse transcription (TPRT) reaction. These minimal RNA requirements suggested that if recombination/DNA repair conjoined the 5' and 3' ends of R2, the result would be a non-autonomous element that could survive as long as autonomous R2 elements supplied the TPRT activity.ResultsA PCR-based survey of 39 Drosophila species aided by genomic sequences from 12 of these species revealed two types of non-autonomous elements. We call these elements SIDEs (for ‘Short Internally Deleted Elements’). The first consisted of a 5' ribozyme and a 3' end of an R2 element as predicted. Variation at the 5' junctions of the R2 SIDE copies was typical for R2 insertions suggesting their propagation by TPRT. The second class of SIDE contained sequences from R1 elements, another non-LTR retrotransposon that inserts into rRNA gene loci. These insertions had an R2 ribozyme immediately upstream of R1 3' end sequences. These hybrid SIDEs were inserted at the R1 site with 14 bp target site duplications typical of R1 insertions suggesting they used the R1 machinery for retrotransposition. Finally, the survey revealed examples of U12 small nuclear (sn)RNA and tRNA sequences at the 5' end of R2 elements suggesting the R2 reverse transcriptase can template jump from the R2 transcript to a second RNA during TPRT.ConclusionsThe R2 SIDE and R2/R1 hybrid SIDEs are rare examples of non-autonomous retrotransposons in the Drosophila genome. Associated non-autonomous elements and in vivo template jumps are two additional characteristics R2 shares with other non-LTR retrotransposons such as mammalian L1s. Analysis of the hybrid SIDEs provides supporting evidence that R1 elements, like R2 elements, recognize their 3' untranslated region (UTR) sequences and, thus, belong to the stringent class of non-LTR elements.

High-valent [MnFe] and [FeFe] cofactors in ribonucleotide reductases

Ribonucleotide reductases (RNRs) are essential for DNA synthesis in most organisms. In class-Ic RNR from Chlamydia trachomatis (Ct), a MnFe cofactor in subunit R2 forms the site required for enzyme activity, instead of an FeFe cofactor plus a redox-active tyrosine in class-Ia RNRs, for example in mouse (Mus musculus, Mm). For R2 proteins from Ct and Mm, either grown in the presence of, or reconstituted with Mn and Fe ions, structural and electronic properties of higher valence MnFe and FeFe sites were determined by X-ray absorption spectroscopy and complementary techniques, in combination with bond-valence-sum and density functional theory calculations. At least ten different cofactor species could be tentatively distinguished. In Ct R2, two different Mn(IV)Fe(III) site configurations were assigned either L4MnIV(μO)2FeIIIL4 (metal–metal distance of ~2.75Å, L = ligand) prevailing in metal-grown R2, or L4MnIV(μO)(μOH)FeIIIL4 (~2.90Å) dominating in metal-reconstituted R2. Specific spectroscopic features were attributed to an Fe(IV)Fe(III) site (~2.55Å) with a L4FeIV(μO)2FeIIIL3 core structure. Several Mn,Fe(III)Fe(III) (~2.9–3.1Å) and Mn,Fe(III)Fe(II) species (~3.3–3.4Å) likely showed 5-coordinated Mn(III) or Fe(III). Rapid X-ray photoreduction of iron and shorter metal–metal distances in the high-valent states suggested radiation-induced modifications in most crystal structures of R2. The actual configuration of the MnFe and FeFe cofactors seems to depend on assembly sequences, bound metal type, valence state, and previous catalytic activity involving subunit R1. In Ct R2, the protonation of a bridging oxide in the MnIV(μO)(μOH)FeIII core may be important for preventing premature site reduction and initiation of the radical chemistry in R1.

The Manganese Ion of the Heterodinuclear Mn/Fe Cofactor in Chlamydia trachomatis Ribonucleotide Reductase R2c Is Located at Metal Position 1

The essential catalytic radical of Class-I ribonucleotide reductase is generated and delivered by protein R2, carrying a dinuclear metal cofactor. A new R2 subclass, R2c, prototyped by the Chlamydia trachomatis protein was recently discovered. This protein carries an oxygen-activating heterodinuclear Mn(II)/Fe(II) metal cofactor and generates a radical-equivalent Mn(IV)/Fe(III) oxidation state of the metal site, as opposed to the tyrosyl radical generated by other R2 subclasses. The metal arrangement of the heterodinuclear cofactor remains unknown. Is the metal positioning specific, and if so, where is which ion located? Here we use X-ray crystallography with anomalous scattering to show that the metal arrangement of this cofactor is specific with the manganese ion occupying metal position 1. This is the position proximal to the tyrosyl radical site in other R2 proteins and consistent with the assumption that the high-valent Mn(IV) species functions as a direct substitute for the tyrosyl radical.

Inhibition of chlamydial class Ic ribonucleotide reductase by C‐terminal peptides from protein R2

Chlamydia trachomatis ribonucleotide reductase (RNR) is a class Ic RNR. It has two homodimeric subunits: proteins R1 and R2. Class Ic protein R2 in its most active form has a manganese-iron metal cofactor, which functions in catalysis like the tyrosyl radical in classical class Ia and Ib RNRs. Oligopeptides with the same sequence as the C-terminus of C. trachomatis protein R2 inhibit the catalytic activity of C. trachomatis RNR, showing that the class Ic enzyme shares a similar highly specific inhibition mechanism with the previously studied radical-containing class Ia and Ib RNRs. The results indicate that the catalytic mechanism of this class of RNRs with a manganese-iron cofactor is similar to that of the tyrosyl-radical-containing RNRs, involving reversible long-range radical transfer between proteins R1 and R2. The competitive binding of the inhibitory R2-derived oligopeptide blocks the transfer pathway. We have constructed three-dimensional structure models of C. trachomatis protein R1, based on homologous R1 crystal structures, and used them to discuss possible binding modes of the peptide to protein R1. Typical half maximal inhibitory concentration values for C. trachomatis RNR are about 200 µ m for a 20-mer peptide, indicating a less efficient inhibition compared with those for an equally long peptide in the Escherichia coli class Ia RNR. A possible explanation is that the C. trachomatis R1/R2 complex has other important interactions, in addition to the binding mediated by the R1 interaction with the C-terminus of protein R2.

Apoptosis induced clustering of IP3R1 in nuclei of non-differentiated PC12 cells

Inositol 1,4,5-trisphosphate (IP(3)) receptors are emerging as key sites for regulation by pro- and anti-apoptotic factors. Induction of apoptosis for 3 h increased mRNA and protein levels of type 1 IP(3) receptors in non-differentiated (ND), but not in differentiated (D) PC12 cells. Inhibitors of the IP(3) R's calcium release-2-aminoethoxydiphenyl borate (2-APB) and xestospongin-completely prevented Bax and caspase-3 mRNA increase after treatment with the apoptosis inducer set (AIK), and this reinforces the importance of IP(3) R1 in the apoptosis of ND PC12 cells. Apoptosis induction not only increases the IP(3) R1 protein, but it also causes formation of IP(3) R1 clusters in the nucleus which most likely result from fusion of the nucleoplasmic reticulum and/or IP(3) R1 translocation to the nucleus. This is quite similar to the observations noted after overexpression of IP(3) R1 in PC12 cells. The amount of IP(3) induced calcium release was higher in control than in AIK-treated cells. From our results we propose that after the apoptosis induction the amount of intranuclear calcium decreased dramatically due to the increase of calcium permeability of the nuclear calcium store vesicles. Therefore, increase of the calcium permeability may result from IP(3) receptors translocation to nuclei that can boost the calcium transport through IP(3) receptors.