SAMHD1, an analogue of the murine interferon (IFN)-γ-induced gene Mg11 (ref. 1), has recently been identified as a human immunodeficiency virus-1 (HIV-1) restriction factor that blocks early-stage virus replication in dendritic and other myeloid cells and is the target of the lentiviral protein Vpx, which can relieve HIV-1 restriction. SAMHD1 is also associated with Aicardi-Goutières syndrome (AGS), an inflammatory encephalopathy characterized by chronic cerebrospinal fluid lymphocytosis and elevated levels of the antiviral cytokine IFN-α. The pathology associated with AGS resembles congenital viral infection, such as transplacentally acquired HIV. Here we show that human SAMHD1 is a potent dGTP-stimulated triphosphohydrolase that converts deoxynucleoside triphosphates to the constituent deoxynucleoside and inorganic triphosphate. The crystal structure of the catalytic core of SAMHD1 reveals that the protein is dimeric and indicates a molecular basis for dGTP stimulation of catalytic activity against dNTPs. We propose that SAMHD1, which is highly expressed in dendritic cells, restricts HIV-1 replication by hydrolysing the majority of cellular dNTPs, thus inhibiting reverse transcription and viral complementary DNA (cDNA) synthesis.

SAMHD1 restricts HIV-1 infection of myeloid-lineage and resting CD4+ T-cells. Most likely this occurs through deoxynucleoside triphosphate triphosphohydrolase activity that reduces cellular dNTP to a level where reverse transcriptase cannot function, although alternative mechanisms have been proposed recently. Here, we present combined structural and virological data demonstrating that in addition to allosteric activation and triphosphohydrolase activity, restriction correlates with the capacity of SAMHD1 to form “long-lived” enzymatically competent tetramers. Tetramer disruption invariably abolishes restriction but has varied effects on in vitro triphosphohydrolase activity. SAMHD1 phosphorylation also ablates restriction and tetramer formation but without affecting triphosphohydrolase steady-state kinetics. However phospho-SAMHD1 is unable to catalyse dNTP turnover under conditions of nucleotide depletion. Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity where dephosphorylation switches housekeeping SAMHD1 found in cycling cells to a high-activity stable tetrameric form that depletes and maintains low levels of dNTPs in differentiated cells.

In order to evaluate further the relationship between acute bronchiolitis in infancy and subsequent respiratory problems, children prospectively followed up from the time of their admission to hospital were reviewed along with a group of matched controls recruited at the previous five and a half year assessment. Sixty one index children and 47 controls took part. The groups were well matched for age, height, parental smoking, and social class. Although the prevalence of respiratory symptoms had fallen when related to the previous review, there remained an excess of coughing (48 and 17% in index and control children respectively; odds ratio 4.02) and wheezing (34 and 13% in index and control children respectively; odds ratio 3.59). Bronchodilator therapy was used by 33% of index children compared with 3% of controls. Lung function tests revealed no significant diVerences in the measurements of lung growth-for example, forced vital capacity, functional residual capacity, and total lung capacity-but the index children had significant reductions in measurements of airways obstruction-for example, forced expiratory volume in one second, maximum expiratory flow at 75, 50 and 25% of vital capacity, and airways resistance. Family history and personal skin tests showed no excess of atopy in the index group. This study supports the claim that the excess respiratory symptoms after acute bronchiolitis are not due to familial or personal susceptibility to atopy.

As part of a long term prospective study, 73 children who had been admitted to hospital with viral bronchiolitis as infants, were reviewed 5-5 years later and compared with a carefully matched control group. In the postbronchiolitis group, there was a highly significant increase in respiratory symptoms including wheezing (42.5% v 15-0%, relative risk=2-8).Although atopy in the family was not significantly increased in the index group, personal atopy was more prevalent. However, personal atopy was not significantly more prevalent in the symptomatic postbronchiolitis, compared with those who were symptom free, and so did not account for the high prevalence of postbronchiolitis wheezing in this cohort. In addition, in a stepwise logistic regressional model, bronchiolitis remained a significant predictor of wheezing after adjusting for potential confounding variables, including atopy. Bronchial responsiveness to histamine was significantly increased in the index group. However, no significant relationship of positive tests to wheezing could be demonstrated, and a high rate of positive responses was noted in the controls.

Germline mutations in the tumor suppressor gene BRCA1 confer an estimated lifetime risk of 56–80% for breast cancer and 15–60% for ovarian cancer. Since the mid 1990’s when BRCA1 was identified, genetic testing has revealed over 3,000 unique germline variants. However, for a significant number of these variants, the effect on protein function is unknown making it difficult to infer the consequences on risks of breast and ovarian cancers. Thus, many individuals undergoing genetic testing for BRCA1 mutations receive test results reporting a variant of uncertain clinical significance (VUS), leading to issues in risk assessment, counseling, and preventive care. Here we describe functional assays for BRCA1 to directly or indirectly assess the impact of a variant on protein conformation or function and how these results can be used to complement genetic data to classify a VUS as to its clinical significance. Importantly, these methods may provide a framework for genome-wide pathogenicity assignment.

Resonance Raman spectra of azurin, a 14.6 kDa Type 1 blue copper protein from Pseudomonas aeruginosa, have been measured at wavelengths throughout the S(Cys) → Cu(II) charge-transfer absorption band centered at 625 nm in an effort to determine the role of environment and structure on the dynamics of excited-state charge transfer. Azurin provides an analogous system to plastocyanin, another Type 1 blue copper protein, whose excited-state structure and dynamics have been previously determined for a number of plant species. Self-consistent analysis of the absorption spectrum and the resulting resonance Raman excitation profiles using a time-dependent wave packet propagation formalism indicates that inhomogeneous effects account for the majority of the spectral broadening of the charge-transfer absorption band, in contrast to the primarily homogeneously broadened charge-transfer absorption band in plastocyanin. The total reorganization energy from the resonance Raman enhanced modes was found to be 0.26 ± 0.02 eV, compared to 0.19 ± 0.02 eV for plastocyanin. A detailed comparison of the copper environment in the two proteins reveals specific differences in structure and hydrogen-bonding environment which may explain the differences in observed excited-state charge-transfer dynamics of azurin and plastocyanin. The X-ray crystal structures of poplar a plastocyanin and P. aeruginosa azurin suggest that the larger coordination number accounts for the increased reorganization energy in azurin, and the increased hydrogen bonding at the copper site and/or conformational substates may explain the greater inhomogeneous component to the absorption line width in azurin.

Highly branched poly(NIPAM) have been prepared using the technique of reversible addition-fragmentation chain transfer (RAFT) polymerisation using a chain transfer agent that allows the incorporation of imidazole functionality in the polymer chain-ends. The lower critical solution temperature (LCST) of the polymers can be controlled by the amount of hydrophobe and GMA incorporated during copolymerisation procedures. These thermally responsive "smart" polymers were used to purify a His-tagged BRCA-1 protein fragment by affinity precipitation. [Diagram: see text]

Three new ruthenium(II) complexes containing the tris(1-pyrazolyl)methane (tpm) ligand have been prepared: [Ru(tpm)(L)(dppn)]n+ (where n = 1; L = Cl (5), n = 2; L = MeCN (6) and pyridine (7); dppn = benzo[i]dipyrido[3,2-a:2',3'-c]phenazine). Complex 6 was structurally characterized by single-crystal X-ray diffraction. Binding parameters of these complexes with calf thymus DNA are reported and compared to those obtained for a previously reported monocation, [RuCl(tpm)(dppz)]+. Binding studies with the dications and the synthetic oligonucleotides poly(dA).poly(dT) and poly(dG).poly(dC) have also been determined. Photophysical and electrochemical properties of 5-7 have been investigated and compared with their dipyridophenazine (dppz) analogues.