A novel autoregulatory loop between the Gcn2-Atf4 pathway and L-Proline metabolism controls stem cell identity

Increasing evidence indicates that metabolism is implicated in the control of stem cell identity. Here, we demonstrate that embryonic stem cell (ESC) behaviour relies on a feedback loop that involves the non-essential amino acid L-Proline (L-Pro) in the modulation of the Gcn2-Eif2α-Atf4 amino acid starvation response (AAR) pathway that in turn regulates L-Pro biosynthesis. This regulatory loop generates a highly specific intrinsic shortage of L-Pro that restricts proliferation of tightly packed domed-like ESC colonies and safeguards ESC identity. Indeed, alleviation of this nutrient stress condition by exogenously provided L-Pro induces proliferation and modifies the ESC phenotypic and molecular identity towards that of mesenchymal-like, invasive pluripotent stem cells. Either pharmacological inhibition of the prolyl-tRNA synthetase by halofuginone or forced expression of Atf4 antagonises the effects of exogenous L-Pro. Our data provide unprecedented evidence that L-Pro metabolism and the nutrient stress response are functionally integrated to maintain ESC identity.Naturally occurring amino acids are emerging as key players in the regulation of the phenotypic plasticity of stem cells.^{1, 2, 3, 4, 5} Indeed, exogenously provided threonine and methionine, two essential amino acids (EAAs), regulate self-renewal and differentiation of pluripotent stem cells.² Moreover, exogenously provided L-Proline (L-Pro), a non-essential amino acid (NEAA), induces mouse ESCs towards an embryonic stem cell-to-mesenchymal-like transition (esMT) that converts compact, adherent ESCs into mesenchymal-like spindle-shaped, highly invasive and metastatic pluripotent stem cells.⁴ This fully reversible process resembles the epithelial-to-mesenchymal transition (EMT), which is essential for normal development and contributes to pathological cancer progression.^{6, 7, 8} Interestingly, the Aldh18a1 gene is specifically induced in and marks the Primitive Endoderm (PrE) in the time window when the pluripotent epiblast precursors are specified within the inner cell mass (ICM) of the blastocyst.⁹ Since the Aldh18a1 enzyme catalyses the first and rate-limiting step of L-Pro biosynthesis, these findings suggest that L-Pro metabolism may regulate cell lineage segregation in early mammalian embryos. Despite its relevance, the molecular mechanisms underlying L-Pro control of stem cell identity remain largely unknown. This prompted us to investigate the early molecular events regulated by exogenously provided L-Pro in mouse ESCs.     

Development of ligands at γ-aminobutyrric acid type A (GABAA) receptor subtype as new agents for pain relief

The identification of compounds with selective anxiolytic-like effects, exerted through the benzodiazepine site on γ-aminobutyric acid type A (GABA_A) receptors, and that show pronounced antihyperalgesia in several pain models, has oriented research towards the development of new agents for the relief of pain. Starting from our previously reported ligands at the benzodiazepine site on GABA_A receptors showing selective anxiolytic-like effects, we have designed new compounds with the aim of identifying those devoid of the typical side effects of the classical benzodiazepines. Our preliminary results indicate that compounds 4, 10(±) and 11 have a very promising antihyperalgesic profile in different animal pain models (peripheral mono-neuropathy, STZ-induced hyperalgesia). In particular 11 exhibits high potency since it exerted its protective effect starting from the dose of 3 mg/kg po, after single injection.     

An aggregation framework to link indicators associated with multifunctional land use to the stakeholder evaluation of policy options

In the last decade efforts have been carried out by the scientific community aimed at building integrated frameworks to support the decision-making process when sustainability issues are addressed. This paper proposes a further advancement in integrated assessment procedures by setting up an operational multi-scale and transparent framework, which comprises the assessment of European regions in terms of sustainability, and the identification of the impact that policy options might have on the sustainability of these regions. The framework is designed for use in ex ante sustainability impact assessment of policy scenarios on multifunctionality of land use and integrates economic, environmental and social issues across a variety of sectors (agriculture, forestry, transport, tourism and energy). The proposed method provides a conceptual framework applicable at different scales (European, regional), and takes into account the great variability of European regions. The described methodology is based on linear additive models to weight and aggregate selected indicators to a set of land use functions identified to describe the goods and services provided by the different land uses that summarise the most relevant economic, environmental and social issues of a region. The framework is designed to allow the evaluation of impacts at an international scale (e.g. the European Union), or on selected regions.The aggregation framework can be used to evaluate the impact that policy options have on the sustainability of multifunctional land use systems with competing demands. A conceptual envelope, called the “trade-off evaluation space”, delineates all possible developments in the functions of the land. The sustainability limits identify the subset of ‘acceptable’ policy options within the trade-off evaluation space, so that the distance of each land use function from sustainability limits can be estimated and trade-offs between the different functions of the multifunctional land use system can be identified. The proposed methodology is adaptable to different contexts: if the assumption is taken that all land use functions are equally weighted the framework can be used to analyse policy cases and take decisions on policy options at the European or regional level. However, at the local-scale the framework can also be applied through a participatory approach and the distribution of weights can be rediscussed with local stakeholders. In both cases the proposed system can be used as a tool for discussion among all interested parties.     

The IL23R R381Q gene variant protects against immune-mediated diseases by impairing IL-23-induced Th17 effector response in humans

IL-23 and Th17 cells are key players in tissue immunosurveillance and are implicated in human immune-mediated diseases. Genome-wide association studies have shown that the IL23R R381Q gene variant protects against psoriasis, Crohn's disease and ankylosing spondylitis. We investigated the immunological consequences of the protective IL23R R381Q gene variant in healthy donors. The IL23R R381Q gene variant had no major effect on Th17 cell differentiation as the frequency of circulating Th17 cells was similar in carriers of the IL23R protective (A) and common (G) allele. Accordingly, Th17 cells generated from A and G donors produced similar amounts of Th17 cytokines. However, IL-23-mediated Th17 cell effector function was impaired, as Th17 cells from A allele carriers had significantly reduced IL-23-induced IL-17A production and STAT3 phosphorylation compared to G allele carriers. Our functional analysis of a human disease-associated gene variant demonstrates that IL23R R381Q exerts its protective effects through selective attenuation of IL-23-induced Th17 cell effector function without interfering with Th17 differentiation, and highlights its importance in the protection against IL-23-induced tissue pathologies.     

Ribosomes deprived of select proteins show similar structural alterations induced by thermal treatment of native particles

Thre different techniques— light scattering, radiowave dielectric spectroscopy, and fluorescence— were employed to investigate conformational variations in Escherichia coli ribosomes induced by removal of specific proteins. To this end, particles were treated with lithium chloride at different ion strength values to produce ribosomal cores. It was previously observed that treatment of ribosomes to subdenaturing temperatures promotes a structural rearrangement that implies a higher exposure of ribosomal RNA to the solvent. Results presented here strongly suggest that protein elimination from the ribosomal particle produces an overall response recalling the same variation of physical parameters previously observed after thermal treatment. We therefore suggest that high salt treatment produces the same structural modification caused by exposure to subdenaturing temperatures.     

Impurity analysis of retinoic acid samples

The structure of an impurity contained in samples of all trans-retinoic acid was established by means of NMR and MS spectra, and confirmed by X-ray diffraction analysis. The chemical structure of the impurity 2 was found to be strictly correlated to the synthetic procedure employed for the preparation of the retinoic acid samples. Single crystal analysis allowed us to characterise the molecular conformation and the crystal structure of 2.     

Relationships of Cysteine and Lysine residues with the substrate binding site of the mitochondrial ornithine/citrulline carrier: an inhibition kinetic approach combined with the analysis of the homology structural model

To gain insights in the relationships of specific amino acid residues with the active site of the mitochondrial ornithine/citrulline carrier, we studied the effect of specific protein modifying reagents on the transport catalysed by the carrier reconstituted into liposomes. It was found that, besides the sulfhydryl reagents NEM, MTSEA, p-hydroxymercuribenzoate, diamide also the lysine reagents PLP, DIDS, SITS, the carboxyl reagents WRK, EDC and the arginine reagent methylglyoxal inhibited the carrier. NEM, MTSEA and PLP inhibited the ornithine/citrulline carrier with a completely competitive type of mechanism. A 1:1 interaction of NEM with the carrier molecule has been demonstrated. The results are in agreement with the localization of one sulfhydryl and at least one amino group in the substrate binding site. On the basis of the interferences between SH reagents and PLP in the transport inhibition, it has been deduced that the distance between the SH and the NH(2) residues of the active site should be comparable to the distance between the gamma-NH(2) and COOH residues of the ornithine molecule. The structural model of the ornithine/citrulline carrier has been obtained by homology modelling using as template the ADP/ATP carrier structure. The combined analysis of the experimental data and the structural model allows to deduce that Cys-132 is located in the substrate binding site, flanked by at least one Lys residue.     

Tyrosine-728 and glutamic acid-735 are essential for the metalloproteolytic activity of the lethal factor of Bacillus anthracis

The lethal factor (LF) of Bacillus anthracis is a Zn2+-endopeptidase specific for the MAPK-kinase family of proteins. The catalytic zinc atom is coordinated by a first shell of residues including the two histidines and the glutamate of the zinc-binding motif HExxH and by Glu-735. A characteristic feature of LF is the presence, within the second shell of residues, of a tyrosine (Tyr-728) in close proximity (3.3 A) to the zinc atom. To investigate the role of Tyr-728 and Glu-735, LF mutants with one or both of these two residues replaced by Ala were cloned, expressed, and purified from Escherichia coli. A fourth mutant was obtained by replacing Tyr-728 with Phe. Spectroscopic analysis of these mutants indicates that they fold in the same way as the parental molecule and that zinc stabilizes the structure of LF. These mutants have neither proteolytic activity nor in vivo toxicity. The possible role of Tyr-728 in catalysis is discussed.