Characterization of the 3':5' ratio for reliable determination of RNA quality

Determination of RNA quality is a critical first step in obtaining meaningful gene expression data. The PCR-based 3′:5′ assay is an RNA quality assessment tool. This assay is a simple, fast, and low-cost method of selecting samples for further analysis. However, its practical applications are unexploited primarily because of the absence of an experimental threshold. We show that, by anchoring the 5′ assay a specific distance from the 3′ end of the sequence and by spacing the 3′ at a distance of a number of nucleotides, a cutoff determines whether a sample is suitable for downstream quantification studies.     

A label-free assay of exonuclease activity using a pyrosequencing technique

Enzymes with 3′-5′ exonuclease activities are important in promoting the accuracy of DNA replication and DNA repair by proofreading. The alteration of the function of these enzymes by endogenous or exogenous effectors could, therefore, have a considerable impact on DNA replication and ultimately on genome integrity. We have developed a label-free high-throughput screening method for quantifying the effects of different reagents on exonuclease activity. The assay is based on a hairpin-forming biotinylated oligonucleotide substrate that contains one or more exonuclease-resistant phosphorothioate nucleotides. The activity and specificity of the selected 3′-5′ exonuclease is determined indirectly using a sensitive pyrosequencing reaction after cleanup of the samples. In this pyrosequencing step, the amount of nucleotides filled into each position of the exonucleolytically degraded 3′ end of the substrate can be recorded quantitatively and equals the amount of the nucleotides removed by the exonuclease. This system allows the estimation of both processivity and efficiency of the exonuclease activity. We have employed compounds reported in the literature to inhibit the exonuclease activities of either exonuclease III or the large fragment of polymerase I (Klenow fragment) to evaluate the assay.     

MCM3AP is transcribed from a promoter within an intron of the overlapping gene for GANP

MCM3 acetylase (MCM3AP) and germinal-centre associated nuclear protein (GANP) are transcribed from the same locus and are therefore confused in databases because the MCM3 acetylase DNA sequence is contained entirely within the much larger GANP sequence and the entire MCM3AP sequence is identical to the carboxy terminus of GANP. Thus, the MCM3AP and GANP genes are read in the same reading frame and MCM3AP is an N-terminally truncated region of GANP. However, we show here that MCM3AP and GANP are different proteins, occupying different locations in the cell and transcribed from different promoters. Intriguingly, a promoter for MCM3AP lies within an intron of GANP. This report is an interesting example in nature of two separate gene products from the same locus that perform two entirely different functions in the cell. Therefore, to avoid further confusion, they should now be referred to as separate but overlapping genes.     

The excision of 3′ penultimate errors by DNA polymerase I and its role in endonuclease V-mediated DNA repair

Deamination of adenine can occur spontaneously under physiological conditions, and is enhanced by exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat, generating the highly mutagenic lesion of deoxyinosine in DNA. Such DNA lesions tends to generate A:T to G:C transition mutations if unrepaired. In Escherichia coli, deoxyinosine is primarily removed through a repair pathway initiated by endonuclease V (endo V). In this study, we compared the repair of three mutagenic deoxyinosine lesions of A-I, G-I, and T-I using E. coli cell-free extracts as well as reconstituted protein system. We found that 3′-5′ exonuclease activity of DNA polymerase I (pol I) was very important for processing all deoxyinosine lesions. To understand the nature of pol I in removing damaged nucleotides, we systemically analyzed its proofreading to 12 possible mismatches 3′-penultimate of a nick, a configuration that represents a repair intermediate generated by endo V. The results showed all mismatches as well as deoxyinosine at the 3′ penultimate site were corrected with similar efficiency. This study strongly supports for the idea that the 3′-5′ exonuclease activity of E. coli pol I is the primary exonuclease activity for removing 3′-penultimate deoxyinosines derived from endo V nicking reaction.     

The X-ray crystal structure of APR-B,an atypical adenosine 5′-phosphosulfate reductase from Physcomitrella patens

Sulfonucleotide reductases catalyse the first reductive step of sulfate assimilation. Their substrate specificities generally correlate with the requirement for a [Fe₄S₄] cluster, where adenosine 5′-phosphosulfate (APS) reductases possess a cluster and 3′-phosphoadenosine 5′-phosphosulfate reductases do not. The exception is the APR-B isoform of APS reductase from the moss Physcomitrella patens, which lacks a cluster. The crystal structure of APR-B, the first for a plant sulfonucleotide reductase, is consistent with a preference for APS. Structural conservation with bacterial APS reductase rules out a structural role for the cluster, but supports the contention that it enhances the activity of conventional APS reductases.     

Crystal Structure of Yeast FAD Synthetase (Fad1) in Complex with FAD

Flavin adenine dinucleotide (FAD) synthetase is an essential enzyme responsible for the synthesis of FAD by adenylation of riboflavin monophosphate (FMN). We have solved the 1.9 Å resolution structure of Fad1, the yeast FAD synthetase, in complex with the FAD product in the active site. The structure of Fad1 shows it to be a member of the PP-ATPase superfamily. Important conformational differences in the two motifs involved in binding the phosphate moieties of FAD compared to the Candida glabrata FMNT ortholog suggests that this loop is dynamic and undergoes substantial conformational changes during its catalytic cycle.     

cNMP-AMs mimic and dissect bacterial nucleotidyl cyclase toxin effects

In addition to the well-known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. The Pseudomonas aeruginosa toxin ExoY massively increases cGMP and cUMP in cells, whereas the Bordetella pertussis toxin CyaA increases cAMP and, to a lesser extent, cCMP. To mimic and dissect toxin effects, we synthesized cNMP-acetoxymethylesters as prodrugs. cNMP-AMs rapidly and effectively released the corresponding cNMP in cells. The combination of cGMP-AM plus cUMP-AM mimicked cytotoxicity of ExoY. cUMP-AM and cGMP-AM differentially activated gene expression. Certain cCMP and cUMP effects were independent of the known cNMP effectors protein kinases A and G and guanine nucleotide exchange factor Epac. In conclusion, cNMP-AMs are useful tools to mimic and dissect bacterial nucleotidyl cyclase toxin effects.     

Astrocytes modulate the chemokine network in a pathogen-specific manner

Immune responses in the central nervous system (CNS) are carefully regulated. Despite the absence of most immune processes and a substantive blood brain barrier, potent immune responses form during infection and autoimmunity. Astrocytes are innate immune sentinels that ensheath parenchymal blood vessels and sit at the gateway to the CNS parenchyma. Viral and bacterial infections trigger the influx of distinct leukocyte subsets. We show that astrocytes alone are sufficient for distinguishing between these two main types of infection and triggers release of relevant chemokines that relate to the microbe recognised. Bacterial-associated molecules induced the preferential expression of CCL2, CXCL1, CCL20 and CCL3 whilst a virus-associated dsRNA analogue preferentially up-regulated CXCL10 and CCL5. Thus, astrocytes can respond to infection in a distinct and appropriate manner suggesting they have the capacity to attract appropriate sets of leukocytes into the brain parenchyma. Astrocytes themselves are unable to respond to these chemokines since they were devoid of most chemokine receptors but expressed CXCR4, CXCR7 and CXCR6 at rest. Stimulation with TGF-β specifically up-regulated CXCR6 expression and may explain how TGF-β/CXCL16-expressing gliomas are so effective at attracting astroglial cells.     

PDE7A1 hydrolyzes cCMP

The degradation and biological role of the cyclic pyrimidine nucleotide cCMP is largely elusive. We investigated nucleoside 3′,5′-cyclic monophosphate (cNMP) specificity of six different recombinant phosphodiesterases (PDEs) by using a highly-sensitive HPLC–MS/MS detection method. PDE7A1 was the only enzyme that hydrolyzed significant amounts of cCMP. Enzyme kinetic studies using purified GST-tagged truncated PDE7A1 revealed a cCMP K_M value of 135 ± 19 μM. The V_max for cCMP hydrolysis reached 745 ± 27 nmol/(min mg), which is about 6-fold higher than the corresponding velocity for adenosine 3′,5′-cyclic monophosphate (cAMP) degradation. In summary, PDE7A is a high-speed and low-affinity PDE for cCMP.     

Structural insight into the mechanism of substrate specificity and catalytic activity of an HD-domain phosphohydrolase: the 5'-deoxyribonucleotidase YfbR from Escherichia coli

HD-domain phosphohydrolases have nucleotidase and phosphodiesterase activities and play important roles in the metabolism of nucleotides and in signaling. We present three 2.1-Å-resolution crystal structures (one in the free state and two complexed with natural substrates) of an HD-domain phosphohydrolase, the Escherichia coli 5′-nucleotidase YfbR. The free-state structure of YfbR contains a large cavity accommodating the metal-coordinating HD motif (H33, H68, D69, and D137) and other conserved residues (R18, E72, and D77). Alanine scanning mutagenesis confirms that these residues are important for activity. Two structures of the catalytically inactive mutant E72A complexed with Co²⁺ and either thymidine-5′-monophosphate or 2′-deoxyriboadenosine-5′-monophosphate disclose the novel binding mode of deoxyribonucleotides in the active site. Residue R18 stabilizes the phosphate on the Co²⁺, and residue D77 forms a strong hydrogen bond critical for binding the ribose. The indole side chain of W19 is located close to the 2′-carbon atom of the deoxyribose moiety and is proposed to act as the selectivity switch for deoxyribonucleotide, which is supported by comparison to YfdR, another 5′-nucleotidase in E. coli. The nucleotide bases of both deoxyriboadenosine-5′-monophosphate and thymidine-5′-monophosphate make no specific hydrogen bonds with the protein, explaining the lack of nucleotide base selectivity. The YfbR E72A substrate complex structures also suggest a plausible single-step nucleophilic substitution mechanism. This is the first proposed molecular mechanism for an HD-domain phosphohydrolase based directly on substrate-bound crystal structures.     

Differential activation of cAMP- and cGMP-dependent protein kinases by cyclic purine and pyrimidine nucleotides

The cyclic purine nucleotides cAMP and cGMP are well-characterized second messengers and activators of PKA and PKG, respectively. In contrast, the functions of the cyclic pyrimidine nucleotides cCMP and cUMP are poorly understood. cCMP induces relaxation of smooth muscle via PKGI, and phosphodiesterases differentially hydrolyze cNMPs. Here, we report that cNMPs differentially activate PKA isoforms and PKGIα. The combination of cCMP with cAMP reduced the EC₅₀ of cAMP for PKA. PKGIα exhibited higher specificity for the cognate cNMP than PKA. Our data support a role of cCMP and cUMP as second messengers.     

Validation of a novel in vitro assay using ultra performance liquid chromatography–mass spectrometry (UPLC/MS) to detect and quantify hydroxylated metabolites of BDE-99 in rat liver microsomes

The purpose of this study was to develop and validate an ultra performance liquid chromatography–mass spectrometry (UPLC/MS) method to investigate the hepatic oxidative metabolism of 2,2′,4,4′,5-pentabromodiphenyl ether (BDE-99), a widely used flame retardant and ubiquitous environmental contaminant. Hydroxylated metabolites were extracted using liquid-to-liquid extraction, resolved on a C₁₈ column with gradient elution and detected by mass spectrometry in single ion recording mode using electrospray negative ionization. The assay was validated for linearity, accuracy, precision, limit of quantification, range and recovery. Calibration curves were linear (R² ≥ 0.98) over a concentration range of 0.010–1.0 μM for 4-OH-2,2′,3,4′,5-pentabromodiphenyl ether (4-OH-BDE-90), 5′-OH-2,2′,4,4′,5-pentabromodiphenyl ether (5′-OH-BDE-99) and 6′-OH-2,2′,4,4′,5-pentabromodiphenyl ether (6′-OH-BDE-99), and a concentration range of 0.0625–12.5 μM for 2,4,5-tribromophenol (2,4,5-TBP). Inter- and intra-day accuracy values ranged from −2.0% to 6.0% and from −7.7% to 7.3%, respectively, and inter- and intra-day precision values ranged from 2.0% to 8.5% and from 2.2% to 8.6% (n = 6), respectively. The limits of quantification were 0.010 μM for 4-OH-BDE-90, 5′-OH-BDE-99 and 6′-OH-BDE-99, and 0.0625 μM for 2,4,5-TBP. Recovery values ranged between 85 and 100% for the four analytes. The validated analytical method was applied to identify and quantify hydroxy BDE-99 metabolites formed in vitro. Incubation of BDE-99 with rat liver microsomes yielded 4-OH-BDE-90 and 6′-OH-BDE-99 as major metabolites and 5′-OH-BDE-99 and 2,4,5-TBP as minor metabolites. To our knowledge, this is the first validated UPLC/MS method to quantify hydroxylated metabolites of PBDEs without the need of derivatization.