Mechanism of 8-amino-7-oxononanoate synthase: spectroscopic, kinetic, and crystallographic studies

8-Amino-7-oxononanoate synthase (also known as 7-keto-8-aminopelargonate synthase, EC 2.3.1.47) is a pyridoxal 5'-phosphate-dependent enzyme which catalyzes the decarboxylative condensation of L-alanine with pimeloyl-CoA in a stereospecific manner to form 8(S)-amino-7-oxononanoate. This is the first committed step in biotin biosynthesis. The mechanism of Escherichia coli AONS has been investigated by spectroscopic, kinetic, and crystallographic techniques. The X-ray structure of the holoenzyme has been refined at a resolution of 1.7 A (R = 18.6%, R(free) = 21. 2%) and shows that the plane of the imine bond of the internal aldimine deviates from the pyridine plane. The structure of the enzyme-product external aldimine complex has been refined at a resolution of 2.0 A (R = 21.2%, R(free) = 27.8%) and shows a rotation of the pyridine ring with respect to that in the internal aldimine, together with a significant conformational change of the C-terminal domain and subtle rearrangement of the active site hydrogen bonding. The first step in the reaction, L-alanine external aldimine formation, is rapid (k(1) = 2 x 10(4) M(-)(1) s(-)(1)). Formation of an external aldimine with D-alanine, which is not a substrate, is significantly slower (k(1) = 125 M(-)(1) s(-)(1)). Binding of D-alanine to AONS is enhanced approximately 2-fold in the presence of pimeloyl-CoA. Significant substrate quinonoid formation only occurs upon addition of pimeloyl-CoA to the preformed L-alanine external aldimine complex and is preceded by a distinct lag phase ( approximately 30 ms) which suggests that binding of the pimeloyl-CoA causes a conformational transition of the enzyme external aldimine complex. This transition, which is inferred by modeling to require a rotation around the Calpha-N bond of the external aldimine complex, promotes abstraction of the Calpha proton by Lys236. These results have been combined to form a detailed mechanistic pathway for AONS catalysis which may be applied to the other members of the alpha-oxoamine synthase subfamily.     

Increased immunogenicity of an anchor-modified tumor-associated antigen is due to the enhanced stability of the peptide/MHC complex: implications for vaccine design

The use of "anchor-fixed" altered peptide ligands is of considerable interest in the development of therapeutic vaccines for cancer and infectious diseases, but the mechanism by which successful altered peptide ligands elicit enhanced immunity is unclear. In this study, we have determined the crystallographic structure of a major tumor rejection Ag, gp100(209-217), in complex with the HLA-A*0201 (HLA-A2) molecule, as well as the structure of a modified version of the peptide which substitutes methionine for threonine at position 2 (T2M; gp100(209-2M)). The T2M-modified peptide, which is more immunogenic in vitro and in vivo, binds HLA-A2 with a approximately 9-fold greater affinity and has a approximately 7-fold slower dissociation rate at physiological temperature. Within the limit of the crystallographic data, the T2M substitution does not alter the structure of the peptide/HLA-A2 complex. Consistent with this finding, in peripheral blood from 95 human subjects, we were unable to identify higher frequencies of T cells specific for either the native or modified peptide. These data strongly support the conclusion that the greater immunogenicity of the gp100(209-2M) peptide is due to the enhanced stability of the peptide/MHC complex, validating the anchor-fixing approach for generating therapeutic vaccine candidates. Thermodynamic data suggest that the enhanced stability of the T2M-modified peptide/HLA-A2 complex is attributable to the increased hydrophobicity of the modified peptide, but the gain due to hydrophobicity is offset considerably by the loss of a hydrogen bond made by the native peptide to the HLA-A2 molecule. Our findings have broad implications for the optimization of current vaccine-design strategies.     

Acid-labile subunit regulation during the early stages of liver regeneration: implications for glucoregulation

The initiation of liver regeneration is regulated by endogenously produced growth factors and cytokines and is accompanied by suppression of growth hormone (GH) binding to hepatocytes. We have demonstrated some of these factors, particularly GH, which modulate acid-labile subunit (ALS) expression in vitro. Consequently, we investigated ALS hepatic mRNA and serum levels in rats for 24 h after partial hepatectomy (PHx). There was a significant suppression of ALS gene expression (approximately 50%, P < 0.005) and serum levels (approximately 30%, P < 0.02) by 12 h in PHx rats relative to controls. Relative to intact animals, hepatic mRNA and serum levels of ALS were suppressed by approximately 60% at 24 h. Similarly, hepatic GH receptor mRNA levels were significantly reduced in PHx animals. Moreover, hepatocytes isolated from PHx animals were less responsive to GH than those from controls. Overall, our results demonstrate that suppression of ALS gene expression and serum levels during liver regeneration relates to lowered hepatic GH sensitivity. Suppressed circulating ALS may alter insulin-like growth factor bioavailability and constitute a mechanism to maintain relatively normal glucoregulation after loss of liver mass.     

Involvement of the insulin-like growth factor binding proteins in the cancer cell response to DNA damage

The complex mechanisms that cells have evolved to meet the challenge of constant exposure to DNA-damaging stimuli, also serve to protect cancer cells from the cytotoxic effects of chemo- and radiotherapy. IGFBPs appear to be involved, directly or indirectly, in some of these protective mechanisms. Activation of p53 is an early response to genotoxic stress, and all six human IGFBP genes have predicted p53 response elements in their promoter and/or intronic regions, at least some of which are functional. IGFBP3 has been extensively characterized as a p53-inducible gene, but in some cases it is suppressed by mutant p53 forms. DNA double-strand breaks (DSBs), induced by radiotherapy and some chemotherapies, potentially lead to apoptotic cell death, senescence, or repair and recovery. DSB damage can be repaired by homologous recombination or non-homologous end-joining (NHEJ), depending on the cell cycle stage, availability of key repair proteins, and other factors. The epidermal growth factor receptor (EGFR) has been implicated in the NHEJ pathway, and EGFR inhibition may inhibit repair, promoting apoptosis and thus improving sensitivity to chemotherapy or radiotherapy. Both IGFBP-3 and IGFBP-6 interact with components of the NHEJ pathway, and IGFBP-3 can facilitate this process through direct interaction with both EGFR and the catalytic subunit of DNA-PK. Cell fate after DNA damage may in part be regulated by the balance between the sphingolipids ceramide and sphingosine-1-phosphate, and IGFBPs can influence the production of both lipids. A better understanding of the involvement of IGFBPs in the DNA damage response in cancer cells may lead to improved methods of sensitizing cancers to DNA-damaging therapies.     

Apelin reduces myocardial reperfusion injury independently of PI3K/Akt and P70S6 kinase

Apelin, the endogenous ligand of the G protein-coupled APJ receptor, is a peptide mediator with emerging regulatory actions in the heart. The aim of the present studies was to explore potential roles of the apelin/APJ system in myocardial ischaemia/reperfusion injury. To determine the cardiac expression of apelin/APJ and potential regulation by acute ischaemic insult, Langendorff perfused rat hearts were subjected to regional ischaemia (left coronary artery occlusion, 35 min) or ischaemia followed by reperfusion (30 min). Apelin and APJ mRNA expression were then determined in ventricular myocardium by rt-PCR. Unlike APJ mRNA expression, which remained unchanged, apelin mRNA was upregulated 2.4 fold in ventricular myocardium from isolated rat hearts undergoing ischaemia alone, but returned back to control levels after 30 min reperfusion. We then proceeded to test the hypothesis that treatment with exogenous apelin is protective against ischaemia/reperfusion injury. Perfused hearts were subjected to 35 min left main coronary artery occlusion and 120 min reperfusion, after which infarct size was determined by tetrazolium staining. Exogenous Pyr(1)-apelin-13 (10(-8 )M) was perfused either from 5 min prior to 15 min after coronary occlusion, or from 5 min prior to 15 min after reperfusion. Whilst ineffective when used during ischaemia alone, apelin administered during reperfusion significantly reduced infarct size (47.6+/-2.6% of ischaemic risk zone compared to 62.6+/-2.8% in control, n=10 each, p<0.05) in hearts subject to temporary coronary occlusion followed by reperfusion. This protective effect was not abolished by co-administration of the PI3K inhibitor wortmannin (10(-7 )M, infarct size 49.8+/-4.1%, n=4) or the P70S6 kinase inhibitor rapamycin (10(-9 )M, 41.8+/-8.8%, n=4). In conclusion these results suggest that apelin may be a new and potentially important cardioprotective autacoid, upregulated rapidly after myocardial ischaemia and acting through an unknown pathway.     

The Burden Attributable to Mental and Substance Use Disorders as Risk Factors for Suicide: Findings from the Global Burden of Disease Study 2010

Background

The Global Burden of Disease Study 2010 (GBD 2010) identified mental and substance use disorders as the 5^th leading contributor of burden in 2010, measured by disability adjusted life years (DALYs). This estimate was incomplete as it excluded burden resulting from the increased risk of suicide captured elsewhere in GBD 2010''s mutually exclusive list of diseases and injuries. Here, we estimate suicide DALYs attributable to mental and substance use disorders.

Methods

Relative-risk estimates of suicide due to mental and substance use disorders and the global prevalence of each disorder were used to estimate population attributable fractions. These were adjusted for global differences in the proportion of suicide due to mental and substance use disorders compared to other causes then multiplied by suicide DALYs reported in GBD 2010 to estimate attributable DALYs (with 95% uncertainty).

Results

Mental and substance use disorders were responsible for 22.5 million (14.8–29.8 million) of the 36.2 million (26.5–44.3 million) DALYs allocated to suicide in 2010. Depression was responsible for the largest proportion of suicide DALYs (46.1% (28.0%–60.8%)) and anorexia nervosa the lowest (0.2% (0.02%–0.5%)). DALYs occurred throughout the lifespan, with the largest proportion found in Eastern Europe and Asia, and males aged 20–30 years. The inclusion of attributable suicide DALYs would have increased the overall burden of mental and substance use disorders (assigned to them in GBD 2010 as a direct cause) from 7.4% (6.2%–8.6%) to 8.3% (7.1%–9.6%) of global DALYs, and would have changed the global ranking from 5^th to 3^rd leading cause of burden.

Conclusions

Capturing the suicide burden attributable to mental and substance use disorders allows for more accurate estimates of burden. More consideration needs to be given to interventions targeted to populations with, or at risk for, mental and substance use disorders as an effective strategy for suicide prevention.     

Genomic organization of gypsy chromatin insulators in Drosophila melanogaster

Chromatin insulators have been implicated in the regulation of higher-order chromatin structure and may function to compartmentalize the eukaryotic genome into independent domains of gene expression. To test this possibility, we used biochemical and computational approaches to identify gypsy-like genomic-binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein, a component of the gypsy insulator. EMSA and FISH analyses suggest that these are genuine Su(Hw)-binding sites. In addition, functional tests indicate that genomic Su(Hw)-binding sites can inhibit enhancer-promoter interactions and thus function as bona fide insulators. The insulator strength is dependent on the genomic location of the transgene and the number of Su(Hw)-binding sites, with clusters of two to three sites showing a stronger effect than individual sites. These clusters of Su(Hw)-binding sites are located mostly in intergenic regions or in introns of large genes, an arrangement that fits well with their proposed role in the formation of chromatin domains. Taken together, these data suggest that genomic gypsy-like insulators may provide a means for the compartmentalization of the genome within the nucleus.     

Analysis of factors that influence energy expenditure in honeyeaters (Meliphagidae)

The energy expenditure of the tūī (Prosthemadera novaeseelandiae), a meliphagid endemic to New Zealand, was measured and compared with 20 species of honeyeaters (family Meliphagidae) to determine whether its expenditure is influenced either by life in a moist, temperate climate or an island residence. Body mass in the honeyeaters accounted for 91.5% of the variation in basal rate. The combination of body mass, climate and the maximal limit to an altitudinal distribution explained 98.6% of the variation in basal rate with tropical, low-altitude species having the highest mass-independent rate. The basal rates of meliphagids in tropical highlands are similar to those in temperate lowlands, which may reflect similar food supplies. The tūī mass-independent expenditure appears to reflect an active lifestyle in a temperate climate with no evidence that an island residence influenced its rate, whereas sedentary birds on New Zealand have responded to island life with a depressed basal rate. An effective analysis of the variation in energy expenditure requires the inclusion of the ecological and behavioural characteristics that distinguish species.     

Determination of metabolic fluxes in a non-steady-state system

Estimation of fluxes through metabolic networks from redistribution patterns of (13)C has become a well developed technique in recent years. However, the approach is currently limited to systems at metabolic steady-state; dynamic changes in metabolic fluxes cannot be assessed. This is a major impediment to understanding the behaviour of metabolic networks, because steady-state is not always experimentally achievable and a great deal of information about the control hierarchy of the network can be derived from the analysis of flux dynamics. To address this issue, we have developed a method for estimating non-steady-state fluxes based on the mass-balance of mass isotopomers. This approach allows multiple mass-balance equations to be written for the change in labelling of a given metabolite pool and thereby permits over-determination of fluxes. We demonstrate how linear regression methods can be used to estimate non-steady-state fluxes from these mass balance equations. The approach can be used to calculate fluxes from both mass isotopomer and positional isotopomer labelling information and thus has general applicability to data generated from common spectrometry- or NMR-based analytical platforms. The approach is applied to a GC-MS time-series dataset of (13)C-labelling of metabolites in a heterotrophic Arabidopsis cell suspension culture. Threonine biosynthesis is used to demonstrate that non-steady-state fluxes can be successfully estimated from such data while organic acid metabolism is used to highlight some common issues that can complicate flux estimation. These include multiple pools of the same metabolite that label at different rates and carbon skeleton rearrangements.