LRH-1 drives colon cancer cell growth by repressing the expression of the CDKN1A gene in a p53-dependent manner

Liver receptor homologue 1 (LRH-1) is an orphan nuclear receptor that has been implicated in the progression of breast, pancreatic and colorectal cancer (CRC). To determine mechanisms underlying growth promotion by LRH-1 in CRC, we undertook global expression profiling following siRNA-mediated LRH-1 knockdown in HCT116 cells, which require LRH-1 for growth and in HT29 cells, in which LRH-1 does not regulate growth. Interestingly, expression of the cell cycle inhibitor p21 (CDKN1A) was regulated by LRH-1 in HCT116 cells. p21 regulation was not observed in HT29 cells, where p53 is mutated. p53 dependence for the regulation of p21 by LRH-1 was confirmed by p53 knockdown with siRNA, while LRH-1-regulation of p21 was not evident in HCT116 cells where p53 had been deleted. We demonstrate that LRH-1-mediated p21 regulation in HCT116 cells does not involve altered p53 protein or phosphorylation, and we show that LRH-1 inhibits p53 recruitment to the p21 promoter, likely through a mechanism involving chromatin remodelling. Our study suggests an important role for LRH-1 in the growth of CRC cells that retain wild-type p53.     

Ecoinvent 3: assessing water use in LCA and facilitating water footprinting

Purpose

Water footprinting and the assessment of water use in life cycle assessment have become of major interest in sustainability assessments. Various initiatives for combining water resource issues with consumption of products and services have been initiated in the last decade. However, comprehensive databases fulfilling the requirements for addressing these issues have been lacking and are necessary to facilitate efficient and consistent assessments of products and services. To this purpose, ecoinvent focused on integrating appropriate water use data into version 3, since previously water use data has been inconsistently reported and some essential flows were missing. This paper describes the structure of the water use data in ecoinvent, how the data has been compiled and the way it can be used for water footprinting.

Methods

The main changes required for proper assessment of water use are the addition of environmental and product flows in order to allow a water balance over each process. This is in accordance with the strict paradigm in ecoinvent 3 to focus on mass balances, which requires the inclusion of water contents of all products (also for e.g. waste water flows), as well as emissions of water to soil, air and various water bodies. Water inputs from air (e.g. rainwater harvesting) is introduced but is not yet used by any activity.

Results and discussion

Ecoinvent version 3.1 consistently includes the relevant flows to address water use in life cycle assessment (LCA) and calculate water footprints on the product level for most processes including uncertainty information. Although some problems regarding data quality and spatial resolution remain, this is an important step forward and can limit efforts for detailed data collection to the most sensitive processes in the product system. With the combination of data on water use and emissions to water for each process, concentration and corresponding water classes can also be calculated and assessed with existing impact assessment methods.

Conclusions

This comprehensive collection of water use data on the process level facilitates the proper assessment of water use within an LCA and water footprints beyond agricultural production. Especially in LCA, but also in tools for eco-design and specific water footprint, this data is essential and leads to a cost-efficient way of assessing consumption choices and product design decisions with full transparency. It enhances the effectiveness of investing in data collection by performing sensitivity analyses using ecoinvent data to identify the most relevant flows and processes.

     

Stability, unfolding, and structural changes of cofactor-free 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase

Stability, unfolding mechanism, spectroscopic, densimetric, and structural characteristics of the oxidatively stable C69S variant (HodC) of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) have been determined by classical and pressure modulation scanning calorimetry (DSC and PMDSC, respectively), circular dichroism (CD) spectroscopy, differential scanning densimetry (DSD), and dynamic light scattering measurements. At 25 degrees C, hexahistidine-tagged HodC has a hydrodynamic radius of 2.3 nm and is characterized by an unusually high degree of alpha-helical structure of approximately 60%, based on deconvolution of CD spectra. The percentage of beta-sheets and -turns is expected to be relatively low in view of its sequence similarity to proteins of the alpha/beta-hydrolase fold superfamily. His6HodC exhibits three-state unfolding (N <--> I <--> D) with an intermediate state I that exhibits at the transition temperature a volume larger than that of the native or denatured state. The intermediate state I is also associated with the highest isothermal expansion coefficient, alphaP, of the three states and exhibits a significantly lower percentage of alpha-helical structure than the native state. The stability difference between the native and intermediate state is rather small which makes I a potential candidate for reactions with various ligands, particularly those having a preference for the apparently preserved beta-type motifs.     

Solid-state 2H NMR spectroscopy of retinal proteins in aligned membranes

Solid-state 2H NMR spectroscopy gives a powerful avenue to investigating the structures of ligands and cofactors bound to integral membrane proteins. For bacteriorhodopsin (bR) and rhodopsin, retinal was site-specifically labeled by deuteration of the methyl groups followed by regeneration of the apoprotein. 2H NMR studies of aligned membrane samples were conducted under conditions where rotational and translational diffusion of the protein were absent on the NMR time scale. The theoretical lineshape treatment involved a static axial distribution of rotating C-C2H3 groups about the local membrane frame, together with the static axial distribution of the local normal relative to the average normal. Simulation of solid-state 2H NMR lineshapes gave both the methyl group orientations and the alignment disorder (mosaic spread) of the membrane stack. The methyl bond orientations provided the angular restraints for structural analysis. In the case of bR the retinal chromophore is nearly planar in the dark- and all-trans light-adapted states, as well upon isomerization to 13-cis in the M state. The C13-methyl group at the "business end" of the chromophore changes its orientation to the membrane upon photon absorption, moving towards W182 and thus driving the proton pump in energy conservation. Moreover, rhodopsin was studied as a prototype for G protein-coupled receptors (GPCRs) implicated in many biological responses in humans. In contrast to bR, the retinal chromophore of rhodopsin has an 11-cis conformation and is highly twisted in the dark state. Three sites of interaction affect the torsional deformation of retinal, viz. the protonated Schiff base with its carboxylate counterion; the C9-methyl group of the polyene; and the beta-ionone ring within its hydrophobic pocket. For rhodopsin, the strain energy and dynamics of retinal as established by 2H NMR are implicated in substituent control of activation. Retinal is locked in a conformation that is twisted in the direction of the photoisomerization, which explains the dark stability of rhodopsin and allows for ultra-fast isomerization upon absorption of a photon. Torsional strain is relaxed in the meta I state that precedes subsequent receptor activation. Comparison of the two retinal proteins using solid-state 2H NMR is thus illuminating in terms of their different biological functions.     

Mtx Toxins Synergize Bacillus sphaericus and Cry11Aa against Susceptible and Insecticide-Resistant Culex quinquefasciatus Larvae

Two mosquitocidal toxins (Mtx) of Bacillus sphaericus, which are produced during vegetative growth, were investigated for their potential to increase toxicity and reduce the expression of insecticide resistance through their interactions with other mosquitocidal proteins. Mtx-1 and Mtx-2 were fused with glutathione S-transferase and produced in Escherichia coli, after which lyophilized powders of these fusions were assayed against Culex quinquefasciatus larvae. Both Mtx proteins showed a high level of activity against susceptible C. quinquefasciatus mosquitoes, with 50% lethal concentrations (LC₅₀) of Mtx-1 and Mtx-2 of 0.246 and 4.13 μg/ml, respectively. The LC₅₀s were 0.406 to 0.430 μg/ml when Mtx-1 or Mtx-2 was mixed with B. sphaericus, and synergy improved activity and reduced resistance levels. When the proteins were combined with a recombinant Bacillus thuringiensis strain that produces Cry11Aa, the mixtures were highly active against Cry11A-resistant larvae and resistance was also reduced. The mixture of two Mtx toxins and B. sphaericus was 10 times more active against susceptible mosquitoes than B. sphaericus alone, demonstrating the influence of relatively low concentrations of these toxins. These results show that, similar to Cyt toxins from B. thuringiensis subsp. israelensis, Mtx toxins can increase the toxicity of other mosquitocidal proteins and may be useful for both increasing the activity of commercial bacterial larvicides and managing potential resistance to these substances among mosquito populations.     

Structural analysis of the 26S proteasome by cryoelectron tomography

The 26S proteasome is the key enzyme of intracellular protein degradation in eukaryotic cells. It is a multisubunit complex of 2.5 MDa confining the proteolytic action to an inner compartment with tightly controlled access. Structural studies of this intriguing molecular machine have been hampered by its intrinsic instability and its dynamics. Here we have used an unconventional approach to obtain a three-dimensional structure of the holocomplex uncompromised by preparation-induced alterations and unbiased by any starting model. We have performed a tomographic reconstruction, followed by averaging over approx. 150 individual reconstructions, of Drosophila 26S proteasomes suspended in a thin layer of amorphous ice.     

The interaction of bioactive peptides with an immobilized phosphatidylcholine monolayer.

The interaction of three bioactive peptides, bombesin, beta-endorphin, and glucagon with a phosphatidylcholine monolayer that was immobilized to porous silica particles and packed into a stainless steel column cartridge, has been studied using dynamic elution techniques. This immobilized lipid monolayer provides a biophysical model system with which to study the binding of peptides to a lipid membrane. In particular, the influence of temperature and methanol concentration on the affinity of each peptide for the immobilized lipid surface was assessed. For all test peptides, nonlinear retention plots were observed at all temperatures that contrasted sharply with the simple linear plots observed for the small unstructured control molecules N-acetyltryptophanamide and diphenylalanine. An analysis of the thermodynamics of the interaction of peptides with the immobilized monolayer was also carried out. The results revealed that while the peptides interacted with the monolayer predominantly through hydrophobic interactions, the relative contribution of DeltaH(assoc)(O) and DeltaS(assoc)(O) to the overall free energy of association was dependent on the temperature and methanol concentration. In particular, it was evident that under most conditions, the binding of the peptides to the immobilized lipid monolayer was enthalpy-driven, i.e., mediated by nonclassical hydrophobic interactions. Significant band-broadening and asymmetric and split peaks were also observed for bombesin, beta-endorphin, and glucagon at different temperatures and methanol concentrations. These changes in affinity and peak shape are consistent with the formation of multiple conformational species during the interaction of these peptides with the lipid monolayer. In addition, the binding behavior of the three test peptides on an n-octylsilica surface that lacked the phospho headgroups of the phospholipid was significantly different from that observed with the immobilized phosphatidylcholine surface, indicating a specificity of interaction between the peptides and the lipid surface. Overall, these experimental results demonstrate that the biomimetic phosphatidylcholine monolayer provides a stable and sensitive system with which to explore the molecular mechanism of peptide conformational changes during membrane interactions.