Phosphorylation-mediated unfolding of a KH domain regulates KSRP localization via 14-3-3 binding

The AU-rich element (ARE)-mediated mRNA-degradation activity of the RNA binding K-homology splicing regulator protein (KSRP) is regulated by phosphorylation of a serine within its N-terminal KH domain (KH1). In the cell, phosphorylation promotes the interaction of KSRP and 14-3-3ζ protein and impairs the ability of KSRP to promote the degradation of its RNA targets. Here we examine the molecular details of this mechanism. We report that phosphorylation leads to the unfolding of the structurally atypical and unstable KH1, creating a site for 14-3-3ζ binding. Using this site, 14-3-3ζ discriminates between phosphorylated and unphosphorylated KH1, driving the nuclear localization of KSRP. 14-3-3ζ –KH1 interaction regulates the mRNA-decay activity of KSRP by sequestering the protein in a separate functional pool. This study demonstrates how an mRNA-degradation pathway is connected to extracellular signaling networks through the reversible unfolding of a protein domain.European Molecular Biology Organization 240-2005Italian CIPE-200

Assessment of a surface-active ionic liquid formulation for EOR applications: Experimental and simulation studies

This study aims to assess a surfactant blend for enhanced oil recovery from carbonate rocks. Due to the abundance of these reservoirs, their profitable exploitation would ensure our petrochemical needs are met, and maintain current quality of life. The objective of this work is to increase the technology readiness level of our previous proposal based on the use of a blend of pure sodium dodecylbenzene sulfonate and the surface-active ionic liquid cocosalkylpentaethoximethyl ammonium methylsulfate. To that aim, the method was adapted for its application with a commercially available petrochemical surfactant (RECOLAS103, a mixture of lineal alkyl benzene sulfonates), and reservoir simulations were carried out to evaluate its effectiveness. Phase behavior, stability, dynamic interfacial tension, adsorption and core flooding were the experimental tests carried out. An optimized formulation consisting of 1 wt% of blend (40 wt% RECOLAS103) in synthetic sea water was found stable and able to reduce water-oil interfacial tension down to 0.02 mN/m. The dynamic blend adsorption in carbonate rocks was found to be 0.60 mg/grock, a promising value for the application. Core flooding tests were conducted at 25 and 120 °C and additional oil recoveries achieved ranged from 10.2 to 12.7% of the original oil in place, the lowest production obtained at the highest temperature. This work offers an advance in the application of surfactants for EOR in carbonate reservoirs, since it improves previous proposals that show stability or high adsorption problems. Moreover, a chemical injection optimization was also carried out by simulation with the CMG-STARS software. Results point to the possibility of reaching higher oil recoveries than those obtained experimentally if the extraction method is optimizedS

Noncanonical G recognition mediates KSRP regulation of let-7 biogenesis

Let-7 is an important tumor-suppressive microRNA (miRNA) that acts as an on-off switch for cellular differentiation and regulates the expression of a set of human oncogenes. Binding of the human KSRP protein to let-7 miRNA precursors positively regulates their processing to mature let-7, thereby contributing to control of cell proliferation, apoptosis and differentiation. Here we analyze the molecular basis for KSRP-let-7 precursor selectivity and show how the third KH domain of the protein recognizes a G-rich sequence in the pre-let-7 terminal loop and dominates the interaction. The structure of the KH3-RNA complex explains the protein recognition of this noncanonical KH target sequence, and we demonstrate that the specificity of this binding is crucial for the functional interaction between the protein and the miRNA precursor

A formulation based on a cationic surface-active ionic liquid and an anionic surfactant for enhanced oil recovery at a carbonate reservoir

With the still huge dependence on crude oil, it is crucial to develop enhanced oil recovery techniques to improve reservoir production and lifespan. Carbonate reservoirs constitute over half the world's oil reserves, but they are challenging in terms of recovery due to their complex pore network, oil-wet or mixed-wet rocks, and harsh conditions of temperature and salinity. This work offers a significant contribution to the exploitation of these reservoirs. A new formulation, able to provide very low interfacial tension at high temperatures with reduced adsorption on carbonate rocks, was designed. A low-cost traditional alkyl benzene sulfonate (RECOLAS158) was mixed with the cationic surface active ionic liquid N,N-diethoxylated-N-tallow-N-ethylammonium ethylsulfate. A formulation containing 56.4 wt% RECOLAS158 was selected for its good performance in terms of phase behavior, injectability and interfacial tension at a wide range of temperatures and salinities. A very significant tertiary oil recovery (19.53% of the original oil in place) and low blend adsorption (0.086 mgblend/grock), demonstrated the promising performance of the formulation. The key mechanism associated to the improvement of oil recovery is interfacial tension reductionThe authors acknowledge the Ministry of Science and Innovation and State Research Agency for financial support throughout project PGC2018-097342-B-I00, including European Regional Development Fund). A. Somoza also acknowledges predoctoral financial support (grant ref. PRE2019-089101)S

NMR Structural Determinants of Eosinophil Cationic Protein Binding to Membrane and Heparin Mimetics

Eosinophil cationic protein (ECP) is a highly stable, cytotoxic ribonuclease with the ability to enter and disrupt membranes that participates in innate immune defense against parasites but also kills human cells. We have used NMR spectroscopy to characterize the binding of ECP to membrane and heparin mimetics at a residue level. We believe we have identified three Arg-rich surface loops and Trp35 as crucial for membrane binding. Importantly, we have provided evidence that the interaction surface of ECP with heparin mimetics is extended with respect to that previously described (fragment 34–38). We believe we have identified new sites involved in the interaction for the first time, and shown that the N-terminal α-helix, the third loop, and the first and last β-strands are key for heparin binding. We have also shown that a biologically active ECP N-terminal fragment comprising the first 45 residues (ECP1–45) retains the capacity to bind membrane and heparin mimetics, thus neither the ECP tertiary structure nor its high conformational stability are required for cytotoxicity

- Structural statistics of the 20 best NMR structures of Ani s 5.

<p>- Structural statistics of the 20 best NMR structures of Ani s 5.</p

Ribbon and surface representations of Ani s 5 with epitope mapping.

<p><b>A</b> and <b>B</b>, Peptide segments representing the minimum unit necessary for IgE/IgG₄ recognition are colored in cyan (E1, 7–18), purple (E2, 19–24), hot pink (E3, 31–36), blue (E4, 40–48), yellow (E7, 79–90), red (E8, 91–102), green (E9, 97–116) and dark green (E11, 109–128). The overlapping region 110–116 is colored in pale green. <b>C</b> and <b>D</b>, Peptide segments recognized simultaneously by three different sera are colored in red (E5, 40–59), green (E6, 85–96) and cyan (E10, 103–122). Two perspectives rotated 180° with respect to each other are shown in each panel.</p

Biophysical characterization of Ani s 5 and effect of divalent cations.

<p><b>A</b>, Far-UV CD spectra of Ani s 5 in the absence (black) or in the presence of different cation concentrations: red, MgCl₂ 2 mM; green, MgCl₂ 20 mM; blue, MgCl₂ 100 mM; cyan, CaCl₂ 100 mM at pH 7. <b>B</b>, Fluorescence emission spectra after excitation at 350 nm of Ani s 5 (black), ANS (red), Ani s 5 with CaCl₂ (dark blue), Ani s 5 with MgCl₂ (yellow), ANS with Ani s 5 (green), ANS with CaCl₂ (cyan), ANS with MgCl₂ (olive green), Ani s 5 with ANS and CaCl₂ (magenta), and Ani s 5 with ANS and MgCl₂ (violet). <b>C</b>, SDS-PAGE of Ani s 5, Ani s 5 with EDTA, 2 mM CaCl₂ or 2 mM MgCl₂ 2. M denotes the molecular weight markers used and the asterisks indicate the 15 kDa standard.</p

Solution structure of Ani s 5.

<p><b>A</b>, Backbone superposition of helices H1–H3 of the 20 lowest-energy conformers of Ani s 5 obtained by NMR. The superimposed helices are in gold and the rest of the protein is in blue. <b>B</b>, Backbone superposition of helices H3–H5 of the 20 lowest-energy conformers of Ani s 5 obtained by NMR. The superimposed helices are in gold and the rest of the protein in blue. The orientation of the ensemble is rotated 180° in <b>A</b> and <b>B</b>. <b>C</b>, Ribbon display of a representative conformer of the family showing the positions of the helical segments (H1 to H5) and one of the possible orientations of the C-terminal tail containing helix H6, with respect to the protein core. Helices H1 (15–27), H2 (33–47), H3 (51–77), H4 (82–96), H5 (103–113), H6 (118–125) and the N- and C-termini are labeled. <b>D</b>, Electrostatic surface of Ani s 5. Negative charges are in red, positive charges are in blue. Circles indicate the hydrophobic regions at the level of H1–H2 and H4–H5 helices. Left, the protein has the same orientation as in A. Right, 180° rotated view.</p