Purine biosynthesis in Escherichia coli K12: structure and DNA sequence studies of the purHD locus

The de novo purine biosynthetic enzymes 5-amino-4-imidazolecarboxamide-ribonucleotide (AICAR) transformylase (EC 2.1.2.3), IMP cyclohydrolase (EC 3.5.4.10) and glycineamide-ribonucleotide (GAR) synthetase (EC 2.1.2.2) are encoded by the purHD locus of Escherichia coli. The DNA sequence of this locus revealed two open reading frames encoding polypeptides of Mr 57,335 and 45,945 (GAR synthetase), respectively, that formed an operon. The DNA sequence, maxicell and complementation analyses all supported the concept that the Mr 57,335 polypeptide is the product of the purH gene and encodes a bifunctional protein containing both AICAR transformylase and IMP cyclohydrolase activities. The 5' end of the purHD mRNA was determined by primer extension mapping and contains two regions of dyad symmetry capable of forming 'hairpin' loops where the formation of the one would prevent the formation of the other but not vice versa. Regulation by the purR gene product was explained by the discovery of a purR binding site in the purHD control region.     

Microsomal and cytosolic cytochrome P450 mediated benzo(a)pyrene hydroxylation in Pleurotus pulmonarius

Summary Microsomal and soluble fractions of Pleurotus pulmonarius exhibited a reduced carbon monoxide difference spectrum with P450 maxima at 448nm and 450–452nm respectively. Substrate induced Type I spectra were observed on addition of benzo(a)pyrene to both fractions. Benzo(a)pyrene hydroxylation was measured using the aryl hydrocarbon hydroxylase assay and was observed to be P450 dependent as indicated by carbon monoxide inhibition together with the substrate binding characteristics. The activity of the fractions were observed to give K_m of 200mM and 660mM and V_max of 1.25 nmol/min/nmol P450 and 0.57 nmol/min/nmol P450 for the microsomal and cytosolic fractions respectively.     

RB signaling prevents replication-dependent DNA double-strand breaks following genotoxic insult

Cell cycle checkpoints induced by DNA damage play an integral role in preservation of genomic stability by allowing cells to limit the propagation of deleterious mutations. The retinoblastoma tumor suppressor (RB) is crucial for the maintenance of the DNA damage checkpoint function because it elicits cell cycle arrest in response to a variety of genotoxic stresses. Although sporadic loss of RB is characteristic of most cancers and results in the bypass of the DNA damage checkpoint, the consequence of RB loss upon chemotherapeutic responsiveness has been largely uninvestigated. Here, we employed a conditional knockout approach to ablate RB in adult fibroblasts. This system enabled us to examine the DNA damage response of adult cells following acute RB deletion. Using this system, we demonstrated that loss of RB disrupted the DNA damage checkpoint elicited by either cisplatin or camptothecin exposure. Strikingly, this bypass was not associated with enhanced repair, but rather the accumulation of phosphorylated H2AX (γH2AX) foci, which indicate DNA double-strand breaks. The formation of γH2AX foci was due to ongoing replication following chemotherapeutic treatment in the RB-deficient cells. Additionally, peak γH2AX accumulation occurred in S-phase cells undergoing DNA replication in the presence of damage, and these γH2AX foci co-localized with replication foci. These results demonstrate that acute RB loss abrogates DNA damage-induced cell cycle arrest to induce γH2AX foci formation. Thus, secondary genetic lesions induced by RB loss have implications for the chemotherapeutic response and the development of genetic instability.     

Mutually cooperative binding of eukaryotic translation initiation factor (eIF) 3 and eIF4A to human eIF4G-1

Eukaryotic translation initiation factor 4G-1 (eIF4G) plays a critical role in the recruitment of mRNA to the 43 S preinitiation complex. The central region of eIF4G binds the ATP-dependent RNA helicase eIF4A, the 40 S binding factor eIF3, and RNA. In the present work, we have further characterized the binding properties of the central region of human eIF4G. Both titration and competition experiments were consistent with a 1:1 stoichiometry for eIF3 binding. Surface plasmon resonance studies showed that three recombinant eIF4G fragments corresponding to amino acids 642-1560, 613-1078, and 975-1078 bound eIF3 with similar kinetics. A dissociation equilibrium constant of approximately 42 nm was derived from an association rate constant of 3.9 x 10(4) m(-1) s(-1) and dissociation rate constant of 1.5 x 10(-3) s(-1). Thus, the eIF3-binding region is included within amino acid residues 975-1078. This region does not overlap with the RNA-binding site, which suggests that eIF3 binds eIF4G directly and not through an RNA bridge, or the central eIF4A-binding site. Surprisingly, the binding of eIF3 and eIF4A to the central region was mutually cooperative; eIF3 binding to eIF4G increased 4-fold in the presence of eIF4A, and conversely, eIF4A binding to the central (but not COOH-terminal) region of eIF4G increased 2.4-fold in the presence of eIF3.     

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.      

Fluorescence Resonance Energy Transfer Analysis of Merlin Conformational Changes

Neurofibromatosis type 2 is an inherited autosomal disorder caused by biallelic inactivation of the NF2 tumor suppressor gene. The NF2 gene encodes a 70-kDa protein, merlin, which is a member of the ezrin-radixin-moesin (ERM) family. ERM proteins are believed to be regulated by a transition between a closed conformation, formed by binding of their N-terminal FERM domain and C-terminal tail domain (CTD), and an open conformation, in which the two domains do not interact. Previous work suggests that the tumor suppressor function of merlin is similarly regulated and that only the closed form is active. Therefore, understanding the mechanisms that control its conformation is crucial. We have developed a series of probes that measures merlin conformation by fluorescence resonance energy transfer, both as purified protein and in live cells. Using these tools, we find that merlin exists predominately as a monomer in a stable, closed conformation that is mediated by the central α-helical domain. The contribution from the FERM-CTD interaction to the closed conformation appears to be less important. Upon phosphorylation or interaction with an effector protein, merlin undergoes a subtle conformational change, suggesting a novel mechanism that modulates the interaction between the FERM domain and the CTD.Neurofibromatosis type 2 is an inherited autosomal disorder that is characterized by bilateral schwannomas of the eighth cranial nerve. The tumor suppressor gene responsible for this disorder, NF2, was cloned in 1993 (45). Biallelic inactivation of the NF2 gene is also seen in spontaneous schwannoma, meningioma, and malignant mesothelioma (22). In mouse models, deletion of the Nf2 gene is embryonic lethal, indicating an essential role for NF2 in development (24). Heterozygous mice develop a variety of aggressive metastatic tumors that have lost the wild-type allele (23). Targeted deletion of the Nf2 gene in Schwann cells leads to schwannoma formation (7). In vitro, Nf2-null cells grow to significantly higher densities (31), suggesting that contact inhibition of growth is impaired in these cells and that mediation of growth arrest at high cell density may be the basis for the tumor suppressor function of the NF2 gene. In normal fibroblasts, merlin is inactive as a growth suppressor in subconfluent cells, becoming activated as they approach confluence, thereby effecting contact inhibition of growth (26).The NF2 gene encodes a 70-kDa protein called merlin (for moesin, ezrin, radixin-like protein), which shares significant homology with members of the ezrin-radixin-moesin (ERM) branch of the Band 4.1 superfamily (45). The domain structure of merlin, also shared with other ERM proteins, consists of an N-terminal FERM domain, followed by a central α-helical region (CH) and a C-terminal tail domain (CTD). The merlin FERM domain has relatively high sequence similarity with other ERM family members, a 60 to 70% identity over the first 300 amino acids. The CH domain and the CTD show much lower identity (28 to 36%); however, the α-helical character of the CH domain is preserved, as is the heptad repeat pattern typical of α-helices that form coiled coils (46).The critical point of regulation of all the ERM proteins is a high-affinity intramolecular interaction between the C-terminal domain and the FERM domain (4) (Fig. ​(Fig.1).1). The FERM domain folds into a three-lobed cloverleaf structure that acts as a multifaceted docking site for protein binding partners (16, 39). The CTD, consisting of four major and two minor helices and a beta sheet, binds to the FERM domain by extending across the face of the F2 and F3 lobes (32). This intramolecular head-to-tail binding results in a “closed” conformation, with the C-terminal domain covering much of the surface of the FERM domain (32, 44). For ezrin, radixin and moesin, the CTD functions as a mask, blocking access of effector molecules, such as the cell surface receptors CD44 and ICAM2 and adaptor molecules, like EBP50/NHERF, to sites on the surface of the FERM domain (11, 25, 44). The interaction between the CTD and FERM domain is regulated by phosphatidyl inositol-(4,5)-bisphosphate (PIP₂) binding to the FERM domain and by phosphorylation of a critical residue in the CTD (3, 6, 10, 49). This residue, threonine 567 in ezrin, is conserved throughout the ERM family (21). Phosphorylation introduces a negative charge and a bulky side group that effectively reduces the affinity of the interaction, releasing the CTD from the FERM domain and causing a transition to an open conformation. Low-angle rotary shadowing electron microscopy (13) and biochemical studies (12) of purified radixin suggest that in the open conformation it is an extended filamentous structure with globular N and C termini that is greater than 240 Å in length. Signal transduction systems, such as the epidermal growth factor and Rho A pathways, induce phosphorylation of ERM proteins at the conserved C-terminal threonine via a number of kinases, including Rho kinase and protein kinase Cα (21, 28). Thus, conformational regulation of ERM proteins can be a point of integration of ERM activity with signal transduction pathways. The overall concept of ERM regulation, then, is centered upon a transition between an inactive, closed conformation that is mediated by the FERM-CTD interaction and an active, open conformation that is regulated by phosphorylation. In these two states, ERM proteins likely interact with different sets of binding partners, resulting in distinct functional outcomes.Open in a separate windowFIG. 1.ERM tertiary structure as represented by the crystal structure of full-length Sf-moesin (20), but with the merlin amino acid sequence substituted for Sf-moesin. Approximate boundary amino acid residues for all domains appear at the top of the figure. Each domain is assigned a different color. The ERM structure consists of an N-terminal FERM domain folded into three lobes, F1, F2, and F3. This is followed by a central α-helical domain containing three subhelices (αA, αB, and αC) and a CTD with four short helices. An ERM protein is thought to have an open conformation, an extended structure with the FERM domain and the CTD separated by the α-helical domain, that is more than 240 Å long. In the closed conformation, the α-helical domain bends at the αA-αB junction and again at the αB-αC junction, causing the CTD to be positioned over F2 and F3 of the FERM domain. More than half of the surface of the FERM domain is masked by interaction with the CTD, αA, and parts of αB and αC.Like the classical ERMs, merlin is also thought to be regulated by changes in conformation. The FERM domain and the CTD of merlin interact with each other, albeit at a lower level of affinity than the ezrin FERM domain and the CTD (29). There are important differences, however, between merlin and the other ERM proteins. First, phosphorylation of the conserved C-tail threonine T576 has not been reported to occur in mammalian merlin, and nonphosphorylatable and phosphomimetic substitutions at this site have no effect on merlin activity (42). Instead, merlin is phosphorylated at serine 518 in the CTD, a target of the p21-activated kinase PAK and protein kinase A (1, 18, 47). The growth-suppressive function of merlin is activated by dephosphorylation of S518 by the phosphatase PP1δ in a density-dependent manner (14). Second, it was reported in a study using FERM domain and CTD truncates of merlin that only cotransfection of both the N-and C-terminal halves resulted in growth suppression (38). Together, these observations suggested a model of inactive, phosphorylated merlin in an open conformation that, upon cell-to-cell contact, is dephosphorylated and transitions to a closed, growth suppressive conformation.The existing model for conformational regulation described above is inferred from indirect data and assays that generally measure the interaction of isolated FERM and CTD truncates rather than full-length molecules (9, 29, 38). It has been impossible to test directly because tools have not been available to specifically assay for either the open or the closed form of merlin. Therefore, we have developed a series of probes that measures merlin conformation by fluorescence resonance energy transfer (FRET), both as purified protein and in live cells. Using these tools, we show that merlin exists predominately as a monomer in a stable, largely closed conformation. Additionally, we find that the closed conformation is largely mediated by the central α-helical domain; the contribution of the FERM-CTD interaction appears to be less significant than previously thought. Finally, we find that phosphorylation and protein interaction cause unexpectedly small changes in merlin conformation. We propose a new and more refined model for merlin regulation, in which merlin function is regulated by specific but subtle conformational changes that modulate the interaction between the FERM domain and the CTD.     

Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.