Crystal structural studies of changes in the native dinuclear iron center of ribonucleotide reductase protein R2 from mouse

Class I ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleotides in mammals and many other organisms. The RNR subunit R2 contains a dinuclear iron center, which in its diferrous form spontaneously reacts with O2, forming a mu-oxo-bridged diferric cluster and a stable tyrosyl radical. Here, we present the first crystal structures of R2 from mouse with its native dinuclear iron center, both under reducing and oxidizing conditions. In one structure obtained under reducing conditions, the iron-bridging ligand Glu-267 adopts the mu-(eta1,eta2) coordination mode, which has previously been related to O2 activation, and an acetate ion from the soaking solution is observed where O2 has been proposed to bind the iron. The structure of mouse R2 under oxidizing conditions resembles the nonradical diferric R2 from Escherichia coli, with the exception of the coordination of water and Asp-139 to Fe1. There are also additional water molecules near the tyrosyl radical site, as suggested by previous spectroscopic studies. Since no crystal structure of the active radical form has been reported, we propose models for the movement of waters and/or tyrosyl radical site when diferric R2 is oxidized to the radical form, in agreement with our previous ENDOR study. Compared with E. coli R2, two conserved phenylalanine residues in the hydrophobic environment around the diiron center have opposing rotameric conformations, and the carboxylate ligands of the diiron center in mouse R2 appear more flexible. Together, this might contribute to the lower affinity and cooperative binding of iron in mouse R2.     

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The C-Terminal Repeating Units of CsgB Direct Bacterial Functional Amyloid Nucleation

Curli are functional amyloids produced by enteric bacteria. The major curli fiber subunit, CsgA, self-assembles into an amyloid fiber in vitro. The minor curli subunit protein, CsgB, is required for CsgA polymerization on the cell surface. Both CsgA and CsgB are composed of five predicted β-strand-loop-β-strand-loop repeating units that feature conserved glutamine and asparagine residues. Because of this structural homology, we proposed that CsgB might form an amyloid template that initiates CsgA polymerization on the cell surface. To test this model, we purified wild-type CsgB and found that it self-assembled into amyloid fibers in vitro. Preformed CsgB fibers seeded CsgA polymerization as did soluble CsgB added to the surface of cells secreting soluble CsgA. To define the molecular basis of CsgB nucleation, we generated a series of mutants that removed each of the five repeating units. Each of these CsgB deletion mutants was capable of self-assembly in vitro. In vivo, membrane-localized mutants lacking the first, second, or third repeating units were able to convert CsgA into fibers. However, mutants missing either the fourth or fifth repeating units were unable to complement a csgB mutant. These mutant proteins were not localized to the outer membrane but were instead secreted into the extracellular milieu. Synthetic CsgB peptides corresponding to repeating units 1, 2, and 4 self-assembled into ordered amyloid polymers, while peptides corresponding to repeating units 3 and 5 did not, suggesting that there are redundant amyloidogenic domains in CsgB. Our results suggest a model where the rapid conversion of CsgB from unstructured protein to a β-sheet-rich amyloid template anchored to the cell surface is mediated by the C-terminal repeating units.     

In vitro complementation of Tdp1 deficiency indicates a stabilized enzyme-DNA adduct from tyrosyl but not glycolate lesions as a consequence of the SCAN1 mutation

A homozygous H493R mutation in the active site of tyrosyl-DNA phosphodiesterase (TDP1) has been implicated in hereditary spinocerebellar ataxia with axonal neuropathy (SCAN1), an autosomal recessive neurodegenerative disease. However, it is uncertain how the H493R mutation elicits the specific pathologies of SCAN1. To address this question, and to further elucidate the role of TDP1 in repair of DNA end modifications and general physiology, we generated a Tdp1 knockout mouse and carried out detailed behavioral analyses as well as characterization of repair deficiencies in extracts of embryo fibroblasts from these animals. While Tdp1^?/? mice appear phenotypically normal, extracts from Tdp1^?/? fibroblasts exhibited deficiencies in processing 3′-phosphotyrosyl single-strand breaks and 3′-phosphoglycolate double-strand breaks (DSBs), but not 3′-phosphoglycolate single-strand breaks. Supplementing Tdp1^?/? extracts with H493R TDP1 partially restored processing of 3′-phosphotyrosyl single-strand breaks, but with evidence of persistent covalent adducts between TDP1 and DNA, consistent with a proposed intermediate-stabilization effect of the SCAN1 mutation. However, H493R TDP1 supplementation had no effect on phosphoglycolate (PG) termini on 3′ overhangs of double-strand breaks; these remained completely unprocessed. Altogether, these results suggest that for 3′-phosphoglycolate overhang lesions, the SCAN1 mutation confers loss of function, while for 3′-phosphotyrosyl lesions, the mutation uniquely stabilizes a reaction intermediate.     

Cocaine produces D2R-mediated conformational changes in the adenosine A2AR-dopamine D2R heteromer

Adenosine A_2A receptors (A_2ARs) and dopamine D₂ receptors (D₂Rs) form constitutive heteromers in living cells and exhibit a strong functional antagonistic interaction. Recent findings give neurochemical evidence that extended cocaine self-administration in the rat give rise to an up-regulation of functional A_2ARs in the nucleus accumbens that return to baseline expression levels during cocaine withdrawal. In the present work, the acute in vitro effects of a concentration of cocaine known to fully block the dopamine (DA) transporter without exerting any toxic actions were investigated on A_2AR and D_2LR formed heteromers in transiently co-transfected HEK-293T cells. In vitro treatment of cocaine was found to produce changes in D₂R homodimers and in A_2AR-D₂R heterodimers detected through bioluminescent energy transfer (BRET). Cocaine was found to produce a time- and concentration-dependent reduction in the BRET_max between A_2AR-D_2LR heterodimers and D_2LR homodimers, but not A_2AR homodimers, indicating its effect on D₂R. Cocaine was evaluated with regard to D₂R binding using a human D_2LR stable expressing CHO cell line and was found to produce an increase in the affinity of hD_2LR for DA. At the level of G protein-coupling, cocaine produced a small, but significant increase in DA-stimulated binding of GTPγS. However, cocaine failed to modulate D₂R agonist-induced inhibition of cAMP in stable hD_2LR CHO cells or the gating of GIRK channels in oocytes. Taken together, these results indicate a direct and specific effect of a moderate concentration of cocaine on the DA D_2LR, that results in enhanced agonist recognition, G protein-coupling and an altered conformational state of D₂R homodimers and A_2AR-D₂R heterodimers.     

The miR-17-92 microRNA cluster regulates multiple components of the TGF-β pathway in neuroblastoma

The miR-17-92 microRNA cluster is often activated in cancer cells, but the identity of its targets remains elusive. Using SILAC and quantitative mass spectrometry, we examined the effects of activation of the miR-17-92 cluster on global protein expression in neuroblastoma (NB) cells. Our results reveal cooperation between individual miR-17-92 miRNAs and implicate miR-17-92 in multiple hallmarks of cancer, including proliferation and cell adhesion. Most importantly, we show that miR-17-92 is a potent inhibitor of TGF-β signaling. By functioning both upstream and downstream of pSMAD2, miR-17-92 activation triggers downregulation of multiple key effectors along the TGF-β signaling cascade as well as direct inhibition of TGF-β-responsive genes.     

Dynamic Dependence on ATR and ATM for Double-Strand Break Repair in Human Embryonic Stem Cells and Neural Descendants

The DNA double-strand break (DSB) is the most toxic form of DNA damage. Studies aimed at characterizing DNA repair during development suggest that homologous recombination repair (HRR) is more critical in pluripotent cells compared to differentiated somatic cells in which nonhomologous end joining (NHEJ) is dominant. We have characterized the DNA damage response (DDR) and quality of DNA double-strand break (DSB) repair in human embryonic stem cells (hESCs), and in vitro-derived neural cells. Resolution of ionizing radiation-induced foci (IRIF) was used as a surrogate for DSB repair. The resolution of γ-H2AX foci occurred at a slower rate in hESCs compared to neural progenitors (NPs) and astrocytes perhaps reflective of more complex DSB repair in hESCs. In addition, the resolution of RAD51 foci, indicative of active homologous recombination repair (HRR), showed that hESCs as well as NPs have high capacity for HRR, whereas astrocytes do not. Importantly, the ATM kinase was shown to be critical for foci formation in astrocytes, but not in hESCs, suggesting that the DDR is different in these cells. Blocking the ATM kinase in astrocytes not only prevented the formation but also completely disassembled preformed repair foci. The ability of hESCs to form IRIF was abrogated with caffeine and siRNAs targeted against ATR, implicating that hESCs rely on ATR, rather than ATM for regulating DSB repair. This relationship dynamically changed as cells differentiated. Interestingly, while the inhibition of the DNA-PKcs kinase (and presumably non-homologous endjoining [NHEJ]) in astrocytes slowed IRIF resolution it did not in hESCs, suggesting that repair in hESCs does not utilize DNA-PKcs. Altogether, our results show that hESCs have efficient DSB repair that is largely ATR-dependent HRR, whereas astrocytes critically depend on ATM for NHEJ, which, in part, is DNA-PKcs-independent.     

Specific haptoglobin expression in bronchoalveolar lavage during differentiation of circulating fibroblast progenitor cells in mild asthma

Haptoglobin is an acute-phase glycoprotein considered to be involved in tissue repair and is produced by fibroblasts and inflammatory cells. By using a gel-based proteomic approach, we show for the first time a possible role for haptoglobin in the differentiation of fibroblast progenitor cells, termed fibrocytes, in patients with mild asthma. Bronchoalveolar lavage fluid (BALF) was performed to sample circulating fibrocytes from patients with mild asthma and nonasthmatic control subjects. Fibrocytes from the airway lumen were characterized by triple staining of the markers CD34/CD45R0/alpha-smooth muscle actin, and subjected to confocal microscopy. The protein expression pattern was analyzed using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF). Elevated levels of haptoglobin expression in BALF was reported in a sub-group of patients with mild asthma (p < 0.05) when compared to the other subjects. In addition, this increase in haptoglobin was accompanied by differentiation of fibrocytes into fibroblast-like cells. When further analyzing the expression pattern of haptoglobin isoforms, a heterozygous expression was detected in the patients where fibrocyte differentiation could be observed. These data raise the possibility that an acute and specific inflammatory state facilitates the differentiation of fibroblast progenitor cells into activated fibroblasts. Furthermore, this study proposes a novel role for haptoglobin in airway remodeling in patients with asthma.     

Hibernation factors directly block ribonucleases from entering the ribosome in response to starvation

Ribosome hibernation is a universal translation stress response found in bacteria as well as plant plastids. The term was coined almost two decades ago and despite recent insights including detailed cryo-EM structures, the physiological role and underlying molecular mechanism of ribosome hibernation has remained unclear. Here, we demonstrate that Escherichia coli hibernation factors RMF, HPF and RaiA (HFs) concurrently confer ribosome hibernation. In response to carbon starvation and resulting growth arrest, we observe that HFs protect ribosomes at the initial stage of starvation. Consistently, a deletion mutant lacking all three factors (ΔHF) is severely inhibited in regrowth from starvation. ΔHF cells increasingly accumulate 70S ribosomes harbouring fragmented rRNA, while rRNA in wild-type 100S dimers is intact. RNA fragmentation is observed to specifically occur at HF-associated sites in 16S rRNA of assembled 70S ribosomes. Surprisingly, degradation of the 16S rRNA 3′-end is decreased in cells lacking conserved endoribonuclease YbeY and exoribonuclease RNase R suggesting that HFs directly block these ribonucleases from accessing target sites in the ribosome.     

BLM helicase-dependent transport of p53 to sites of stalled DNA replication forks modulates homologous recombination

Diverse functions, including DNA replication, recombination and repair, occur during S phase of the eukaryotic cell cycle. It has been proposed that p53 and BLM help regulate these functions. We show that p53 and BLM accumulated after hydroxyurea (HU) treatment, and physically associated and co-localized with each other and with RAD51 at sites of stalled DNA replication forks. HU-induced relocalization of BLM to RAD51 foci was p53 independent. However, BLM was required for efficient localization of either wild-type or mutated (Ser15Ala) p53 to these foci and for physical association of p53 with RAD51. Loss of BLM and p53 function synergistically enhanced homologous recombination frequency, indicating that they mediated the process by complementary pathways. Loss of p53 further enhanced the rate of spontaneous sister chromatid exchange (SCE) in Bloom syndrome (BS) cells, but not in their BLM-corrected counterpart, indicating that involvement of p53 in regulating spontaneous SCE is BLM dependent. These results indicate that p53 and BLM functionally interact during resolution of stalled DNA replication forks and provide insight into the mechanism of genomic fidelity maintenance by these nuclear proteins.