A T/C polymorphism in the GPX4 3′UTR affects the selenoprotein expression pattern and cell viability in transfected Caco-2 cells

Background

Synthesis of selenoproteins such as glutathione peroxidases (GPx) requires a specific tRNA and a stem-loop structure in the 3′untranslated region (3′UTR) of the mRNA. A common single nucleotide polymorphism occurs in the GPX4 gene in a region corresponding to the 3′UTR.

Methods

The two variant 3′UTR sequences were linked to sequences from a selenoprotein reporter gene (iodothyronine deiodinase) and expressed in Caco-2 cells. Clones expressing comparable levels of deiodinase (assessed by real-time PCR) were selected and their response to tert-butyl hydroperoxide assessed by cell viability and measurement of reactive oxygen species. Selenoprotein expression was assessed by real-time PCR, enzyme activity and immunoassay.

Results

When selenium supply was low, cells overexpressing the C variant 3′UTR showed lower viability after oxidative challenge, increased levels of reactive oxygen species and lower GPx activity and SelH mRNA expression compared to cells overexpressing the T variant. After selenium supplementation, cell viability and GPx4 expression were higher in the cells overexpressing the C variant. Expression of transgenes incorporating the T/C variant GPX4 (rs713041) sequences in Caco-2 cells leads to alterations in both cell viability after an oxidative challenge and selenoprotein expression. This suggests that the two variants compete differently in the selenoprotein hierarchy.

General Significance

The data provide evidence that the T/C variant GPX4 (rs713041) alters the pattern of selenoprotein synthesis if selenium intake is low. Further work is required to assess the impact on disease susceptibility.     

Seed reserve composition in 19 tree species of a tropical deciduous forest in Mexico and its relationship to seed germination and seedling growth

Background and Aims

The size and composition of seed reserves may reflect the ecological strategy and evolutionary history of a species and also temporal variation in resource availability. The seed mass and composition of seed reserves of 19 co-existing tree species were studied, and we examined how they varied among species in relation to germination and seedling growth rates, as well as between two years with contrasting precipitation (652 and 384 mm).

Methods

Seeds were collected from a tropical deciduous forest in the northwest of Mexico (Chamela Biological Station). The seed dry mass, with and without the seed coat, and the concentrations of lipids, nitrogen and non-structural carbohydrates for the seed minus seed coat were determined. The anatomical localization of these reserves was examined using histochemical analysis. The germination capacity, rate and lag time were determined. The correlations among these variables, and their relationship to previously reported seedling relative growth rates, were evaluated with and without phylogenetic consideration.

Key Results

There were interannual differences in seed mass and reserve composition. Seed was significantly heavier after the drier year in five species. Nitrogen concentration was positively correlated with seed coat fraction, and was significantly higher after the drier year in 12 species. The rate and lag time of germination were negatively correlated with each other. These trait correlations were also supported for phylogenetic independent contrasts. Principal component analysis supported these correlations, and indicated a negative association of seedling relative growth rate with seed size, and a positive association of germination rate with nitrogen and lipid concentrations.

Conclusions

Nitrogen concentration tended to be higher after the drier year and, while interannual variations in seed size and reserve composition were not sufficient to affect interspecific correlations among seed and seedling traits, some of the reserves were related to germination variables and seedling relative growth rate.     

Leukemia inhibitory factor regulates trafficking of T-type Ca2+ channels

Neuropoietic cytokines such as ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) stimulate the functional expression of T-type Ca(2+) channels in developing sensory neurons. However, the molecular and cellular mechanisms involved in the cytokine-evoked membrane expression of T-type Ca(2+) channels are not fully understood. In this study we investigated the role of LIF in promoting the trafficking of T-type Ca(2+) channels in a heterologous expression system. Our results demonstrate that transfection of HEK-293 cells with the rat green fluorescent protein (GFP)-tagged T-type Ca(2+) channel α(1H)-subunit resulted in the generation of transient Ca(2+) currents. Overnight treatment of α(1H)-GFP-transfected cells with LIF caused a significant increase in the functional expression of T-type Ca(2+) channels as indicated by changes in current density. LIF also evoked a significant increase in membrane fluorescence compared with untreated cells. Disruption of the Golgi apparatus with brefeldin A inhibited the stimulatory effect of LIF, indicating that protein trafficking regulates the functional expression of T-type Ca(2+) channels. Trafficking of α(1H)-GFP was also disrupted by cotransfection of HEK-293 cells with the dominant-negative form of ADP-ribosylation factor (ARF)1 but not ARF6, suggesting that ARF1 regulates the LIF-evoked membrane trafficking of α(1H)-GFP subunits. Trafficking of T-type Ca(2+) channels required transient activation of the JAK and ERK signaling pathways since stimulation of HEK-293 cells with LIF evoked a considerable increase in the phosphorylation of the downstream JAK targets STAT3 and ERK. Pretreatment of HEK-293 cells with the JAK inhibitor P6 or the ERK inhibitor U0126 blocked ERK phosphorylation. Both P6 and U0126 also inhibited the stimulatory effect of LIF on T-type Ca(2+) channel expression. These findings demonstrate that cytokines like LIF promote the trafficking of T-type Ca(2+) channels.     

Overexpression of HEMA1 encoding glutamyl-tRNA reductase

5-Aminolevulinic acid (ALA) synthesis has been shown to be the rate limiting step of tetrapyrrole biosynthesis. Glutamyl-tRNA reductase (GluTR) is the first committed enzyme of plant ALA synthesis and is controlled by interacting regulators, such as heme and the FLU protein. Induced inactivation of the HEMA1 gene encoding GluTR by RNAi expression in tobacco resulted in a reduced activity of Mg chelatase and Fe chelatase indicating a feed-forward regulatory mechanism that links ALA synthesis posttranslationally with late enzymes of tetrapyrrole biosynthesis (Hedtke et al., 2007). Here, the regulatory impact of GluTR was investigated by overexpression of AtHEMA1 in Arabidopsis and tobacco plants. Light-dependent ALA synthesis cannot benefit from an up to 7-fold induced expression of GluTR in Arabidopsis. While constitutive AtHEMA1 overexpression in tobacco stimulates ALA synthesis by 50-90% during light-exposed growth of seedlings, no increase in heme and chlorophyll contents is observed. HEMA1 overexpression in etiolated and dark-grown Arabidopsis and tobacco seedlings leads to additional accumulation of protochlorophyllide. As excessive accumulation of GluTR does not correlate with increased ALA formation, it is hypothesized that ALA synthesis is additionally limited by other effectors that balance the allocation of ALA with the activity of enzymes of chlorophyll and heme biosynthesis.     

A General Scheme to Predict Partner Control Mechanisms in Pairwise Cooperative Interactions Between Unrelated Individuals

Recent years have seen an explosion in the diversity of partner control mechanisms hypothesised to stabilise cooperative behaviour among unrelated individuals. Game theory suggests numerous strategies, each with specific decision rules that allow cooperators to control a non‐contributing partner. This diversity of hypothetical strategies seems likely to reflect diversity in the types of intraspecific cooperation and interspecific mutualism that exist in nature. It is therefore important to provide a framework that explains similarities and differences between the various hypothetical strategies and that predicts how key parameters that describe the natural history of natural systems favour different control mechanisms. We develop a novel unifying framework for pairwise interactions between unrelated individuals, in which we link specific control mechanisms to specific game structures. The latter are defined by unique combinations of the states of five parameters that describe investment, aspects of the payoff matrix, the number of interactions and partner choice. We find that specific control mechanisms potentially have utility in a limited number of game structures; conversely, each game structure may typically offer a few competing control mechanisms. Our framework offers theoreticians specific problems that await mathematical exploration, while at the same time offering empiricists guidelines for evaluating the game structure and corresponding control mechanisms in their systems.     

Metalloprotease meprin beta generates nontoxic N-terminal amyloid precursor protein fragments in vivo

Identification of physiologically relevant substrates is still the most challenging part in protease research for understanding the biological activity of these enzymes. The zinc-dependent metalloprotease meprin β is known to be expressed in many tissues with functions in health and disease. Here, we demonstrate unique interactions between meprin β and the amyloid precursor protein (APP). Although APP is intensively studied as a ubiquitously expressed cell surface protein, which is involved in Alzheimer disease, its precise physiological role and relevance remain elusive. Based on a novel proteomics technique termed terminal amine isotopic labeling of substrates (TAILS), APP was identified as a substrate for meprin β. Processing of APP by meprin β was subsequently validated using in vitro and in vivo approaches. N-terminal APP fragments of about 11 and 20 kDa were found in human and mouse brain lysates but not in meprin β(-/-) mouse brain lysates. Although these APP fragments were in the range of those responsible for caspase-induced neurodegeneration, we did not detect cytotoxicity to primary neurons treated by these fragments. Our data demonstrate that meprin β is a physiologically relevant enzyme in APP processing.     

The Aspergillus fumigatus sialidase is a 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid hydrolase (KDNase): structural and mechanistic insights

Aspergillus fumigatus is a filamentous fungus that can cause severe respiratory disease in immunocompromised individuals. A putative sialidase from A. fumigatus was recently cloned and shown to be relatively poor in cleaving N-acetylneuraminic acid (Neu5Ac) in comparison with bacterial sialidases. Here we present the first crystal structure of a fungal sialidase. When the apo structure was compared with bacterial sialidase structures, the active site of the Aspergillus enzyme suggested that Neu5Ac would be a poor substrate because of a smaller pocket that normally accommodates the acetamido group of Neu5Ac in sialidases. A sialic acid with a hydroxyl in place of an acetamido group is 2-keto-3-deoxynononic acid (KDN). We show that KDN is the preferred substrate for the A. fumigatus sialidase and that A. fumigatus can utilize KDN as a sole carbon source. A 1.45-Å resolution crystal structure of the enzyme in complex with KDN reveals KDN in the active site in a boat conformation and nearby a second binding site occupied by KDN in a chair conformation, suggesting that polyKDN may be a natural substrate. The enzyme is not inhibited by the sialidase transition state analog 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) but is inhibited by the related 2,3-didehydro-2,3-dideoxy-d-glycero-d-galacto-nonulosonic acid that we show bound to the enzyme in a 1.84-Å resolution crystal structure. Using a fluorinated KDN substrate, we present a 1.5-Å resolution structure of a covalently bound catalytic intermediate. The A. fumigatus sialidase is therefore a KDNase with a similar catalytic mechanism to Neu5Ac exosialidases, and this study represents the first structure of a KDNase.     

Lipoprotein(a) levels, apo(a) isoform size, and coronary heart disease risk in the Framingham Offspring Study

The aim of this study was to assess the independent contributions of plasma levels of lipoprotein(a) (Lp(a)), Lp(a) cholesterol, and of apo(a) isoform size to prospective coronary heart disease (CHD) risk. Plasma Lp(a) and Lp(a) cholesterol levels, and apo(a) isoform size were measured at examination cycle 5 in subjects participating in the Framingham Offspring Study who were free of CHD. After a mean follow-up of 12.3 years, 98 men and 47 women developed new CHD events. In multivariate analysis, the hazard ratio of CHD was approximately two-fold greater in men in the upper tertile of plasma Lp(a) levels, relative to those in the bottom tertile (P < 0.002). The apo(a) isoform size contributed only modestly to the association between Lp(a) and CHD and was not an independent predictor of CHD. In multivariate analysis, Lp(a) cholesterol was not significantly associated with CHD risk in men. In women, no association between Lp(a) and CHD risk was observed. Elevated plasma Lp(a) levels are a significant and independent predictor of CHD risk in men. The assessment of apo(a) isoform size in this cohort does not add significant information about CHD risk. In addition, the cholesterol content in Lp(a) is not a significant predictor of CHD risk.     

Evolution of human immunodeficiency virus type 1 in a patient with cross-reactive neutralizing activity in serum

Analysis of longitudinally obtained HIV-1 env sequences from an individual with reported cross-reactive neutralizing activity revealed that the majority of viral variants obtained from serum between 4 and 7 years after seroconversion were unable to persist in peripheral blood. Here we show that these viral variants were more sensitive to autologous serum neutralization, had shorter envelopes with fewer potential N-linked glycosylation sites, and showed lower replication kinetics than successfully evolving HIV-1 variants. These data reflect the host selection pressures on phenotypic characteristics of HIV-1 and illustrate in detail the dynamic interaction between HIV-1 and its host's humoral immune responses.     

RIG-I-mediated antiviral signaling is inhibited in HIV-1 infection by a protease-mediated sequestration of RIG-I

The rapid induction of type I interferon (IFN) is essential for establishing innate antiviral responses. During infection, cytoplasmic viral RNA is sensed by two DExD/H box RNA helicases, RIG-I and MDA5, ultimately driving IFN production. Here, we demonstrate that purified genomic RNA from HIV-1 induces a RIG-I-dependent type I IFN response. Both the dimeric and monomeric forms of HIV-1 were sensed by RIG-I, but not MDA5, with monomeric RNA, usually found in defective HIV-1 particles, acting as a better inducer of IFN than dimeric RNA. However, despite the presence of HIV-1 RNA in the de novo infection of monocyte-derived macrophages, HIV-1 replication did not lead to a substantial induction of IFN signaling. We demonstrate the existence of an evasion mechanism based on the inhibition of the RIG-I sensor through the action of the HIV-1 protease (PR). Indeed, the ectopic expression of PR resulted in the inhibition of IFN regulatory factor 3 (IRF-3) phosphorylation and decreased expression of IFN and interferon-stimulated genes. A downregulation of cytoplasmic RIG-I levels occurred in cells undergoing a single-cycle infection with wild-type provirus BH10 but not in cells transfected with a protease-deficient provirus, BH10-PR(-). Cellular fractionation and confocal microscopy studies revealed that RIG-I translocated from the cytosol to an insoluble fraction during the de novo HIV-1 infection of monocyte-derived macrophages, in the presence of PR. The loss of cytoplasmic RIG-I was prevented by the lysosomal inhibitor E64, suggesting that PR targets RIG-I to the lysosomes. This study reveals a novel PR-dependent mechanism employed by HIV-1 to counteract the early IFN response to viral RNA in infected cells.