Search

The nociceptin/orphanin FQ (N/OFQ)/N/OFQ receptor (NOP) system controls different biological functions including pain and cough reflex. Mixed NOP/opioid receptor agonists elicit similar effects to strong opioids but with reduced side effects. In this work, 31 peptides with the general sequence [Tyr/Dmt1,Xaa5]N/OFQ(1-13)-NH2 were synthesized and pharmacologically characterized for their action at human recombinant NOP/opioid receptors. The best results in terms of NOP versus mu opioid receptor potency were obtained by substituting both Tyr1 and Thr5 at the N-terminal portion of N/OFQ(1-13)-NH2 with the noncanonical amino acid Dmt. [Dmt1,5]N/OFQ(1-13)-NH2 has been identified as the most potent dual NOP/mu receptor peptide agonist so far described. Experimental data have been complemented by in silico studies to shed light on the molecular mechanisms by which the peptide binds the active form of the mu receptor. Finally, the compound exerted antitussive effects in an in vivo model of cough.

Short Linear Motifs (SLiMs) are functional protein microdomains that typically mediate interactions between a short linear region in one protein and a globular domain in another. Surface Plasmon Resonance assays have been performed to determine the binding affinity between PDZ domain of wild type human PALS1 protein and tetradecapeptides representing the SLiMs sequences of SARS-CoV-1 and SARS-CoV-2 E proteins (E-SLiMs). SARS-CoV-2 E-SLiM binds to the human target protein with a higher affinity compared to SARS-CoV-1, showing a difference significantly greater than previously reported using the F318W mutant of PALS1 protein and shorter target peptides. Moreover, molecular dynamics simulations have provided clear evidence of the structural determinants driving this binding process. Specifically, the Arginine 69 residue in the SARS-CoV-2 E-SLiM is the key residue able to both enhance the specific polar interaction with negatively charged pockets of the PALS1 PDZ domain and reduce significantly the mobility of the viral peptide. These experimental and computational data are reinforced by the comparison of the interaction between the PALS1 PDZ domain with the natural ligand CRB1, as well as the corresponding E-SLiMs of other coronavirus members such as MERS and OCF43. Our results provide a model at the molecular level of the strategies used to mimic the endogenous SLiM peptide in the binding of the tight junctions of the host cell, explaining one of the possible reasons of the severity of the infection and pulmonary inflammation by SARS-CoV-2.

The Envelope (E) protein of SARS-CoV-2 is the most enigmatic protein among the four structural ones. Most of its current knowledge is based on the direct comparison to the SARS E protein, initially mistakenly undervalued and subsequently proved to be a key factor in the ER-Golgi localization and in tight junction disruption. We compared the genomic sequences of E protein of SARS-CoV-2, SARS-CoV and the closely related genomes of bats and pangolins obtained from the GISAID and GenBank databases. When compared to the known SARS E protein, we observed a significant difference in amino acid sequence in the C-terminal end of SARS-CoV-2 E protein. Subsequently, in silico modelling analyses of E proteins conformation and docking provide evidences of a strengthened binding of SARS-CoV-2 E protein with the tight junction-associated PALS1 protein. Based on our computational evidences and on data related to SARS-CoV, we believe that SARS-CoV-2 E protein interferes more stably with PALS1 leading to an enhanced epithelial barrier disruption, amplifying the inflammatory processes, and promoting tissue remodelling. These findings raise a warning on the underestimated role of the E protein in the pathogenic mechanism and open the route to detailed experimental investigations.

The conversion of the prion protein (PrP(C)) into prions plays a key role in transmissible spongiform encephalopathies. Despite the importance for pathogenesis, the mechanism of prion formation has escaped detailed characterization due to the insoluble nature of prions. PrP(C) interacts with copper through octarepeat and non-octarepeat binding sites. Copper coordination to the non-octarepeat region has garnered interest due to the possibility that this interaction may impact prion conversion. We used X-ray absorption spectroscopy to study copper coordination at pH 5.5 and 7.0 in human PrP(C) constructs, either wild-type (WT) or carrying pathological mutations. We show that mutations and pH cause modifications of copper coordination in the non-octarepeat region. In the WT at pH 5.5, copper is anchored to His96 and His111, while at pH 7 it is coordinated by His111. Pathological point mutations alter the copper coordination at acidic conditions where the metal is anchored to His111. By using in vitro approaches, cell-based and computational techniques, we propose a model whereby PrP(C) coordinating copper with one His in the non-octarepeat region converts to prions at acidic condition. Thus, the non-octarepeat region may act as the long-sought-after prion switch, critical for disease onset and propagation.

This paper describes the scientific aims and potentials as well as the preliminary technical design of RUDE, an innovative tool for multi-disciplinary investigations in a wide field of scientific, technological and industrial applications. IRIDE will be a high intensity "particles factory", based on a combination of high duty cycle radio-frequency superconducting electron linacs and of high energy lasers. Conceived to provide unique research possibilities for particle physics, for condensed matter physics, chemistry and material science, for structural biology and industrial applications, IRIDE will open completely new research possibilities and advance our knowledge in many branches of science and technology. [RIDE is also supposed to be realized in subsequent stages of development depending on the assigned priorities.

Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, mainly localized at nucleoli, that plays a number of functions in ribosome biogenesis and export, cell cycle control, and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia; mutations map to the C-terminal domain of the protein and cause its denaturation and aberrant cytoplasmic translocation. NPM1 C-terminal domain binds G-quadruplex regions at ribosomal DNA and at gene promoters, including the well characterized sequence from the nuclease-hypersensitive element III region of the c-MYC promoter. These activities are lost by the leukemic variant. Here we analyze the NPM1/G-quadruplex interaction, focusing on residues belonging to both the NPM1 terminal three-helix bundle and a lysine-rich unstructured tail, which has been shown to be necessary for high affinity recognition. We performed extended site-directed mutagenesis and measured binding rate constants through surface plasmon resonance analysis. These data, supported by molecular dynamics simulations, suggest that the unstructured tail plays a double role in the reaction mechanism. On the one hand, it facilitates the formation of an encounter complex through long range electrostatic interactions; on the other hand, it directly contacts the G-quadruplex scaffold through multiple and transient electrostatic interactions, significantly enlarging the contact surface.

In the opportunistic pathogen Pseudomonas aeruginosa the denitrification process is triggered by nitric oxide (NO) and plays a crucial role for the survival in chronic infection sites as a microaerobic-anaerobic biofilm. This respiratory pathway is transcriptionally induced by DNR, an heme-based gas sensor which positively responds to NO. Molecular details of the NO sensing mechanism employed by DNR are now emerging: we recently reported an in vitro study which dissected, for the first time, the heme-iron environment and identified one of the heme axial ligand (i.e. His187), found to be crucial to respond to NO. Nevertheless, the identification of the second heme axial ligand has been unsuccessful, given that a peculiar phenomenon of ligand switching around the heme-iron presumably occurs in DNR. The unusual heme binding properties of DNR could be due to the remarkable flexibility in solution of DNR itself, which, in turns, is crucial for the sensing activity; protein flexibility and dynamics indeed represent a common strategy employed by heme-based redox sensors, which present features deeply different from those of "canonical" hemeproteins. The capability of DNR to deeply rearrange around the heme-iron as been here demonstrated by means of spectroscopic characterization of the H167A/H187A DNR double mutant, which shows unusual kinetics of binding of NO and CO. Moreover, we show that the alteration (such as histidines mutations) of the distal side of the heme pocket is perceived by the proximal one, possibly via the DNR protein chain.

Prion diseases are a class of fatal neurodegenerative disorders characterized by brain spongiosis, synaptic degeneration, microglia and astrocytes activation, neuronal loss and altered redox control. These maladies can be sporadic, iatrogenic and genetic. The etiological agent is the prion, a misfolded form of the cellular prion protein, PrPC. PrPC interacts with metal ions, in particular copper and zinc, through the octarepeat and non-octarepeat binding sites. The physiological implication of this interaction is still unclear, as is the role of metals in the conversion. Since prion diseases present metal dys-homeostasis and increased oxidative stress, we described the copper-binding site located in the human C-terminal domain of PrP - HuPrP(90-231) - both in the wild-type protein and in the protein carrying the pathological mutation Q212P. We used the synchrotron-based X-ray absorption fine structure technique to study the Cu(II) and Cu(I) coordination geometries in the mutant, and we compared them with those obtained using the wild-type protein. By analyzing the extended X-ray absorption fine structure and the X-ray absorption near-edge structure, we highlighted changes in copper coordination induced by the point mutation Q212P in both oxidation states. While in the wild-type protein the copper-binding site has the same structure for both Cu(II) and Cu(I), in the mutant the coordination site changes drastically from the oxidized to the reduced form of the copper ion. Copper-binding sites in the mutant resemble those obtained using peptides, confirming the loss of short- and long-range interactions. These changes probably cause alterations in copper homeostasis and, consequently, in redox control.

We report the first quantitative analysis of the Fe K-edge polarized x-ray absorption near edge structure of the iron protein carbonmonoxy-myoglobin (MbCO) single crystal and of its cryogenic photoproduct Mb(*)CO. The CO-Fe-heme local structure has been determined using a novel fitting procedure based on the full multiple scattering approach. The extracted local structure of Mb(*)CO includes a Fe-CO distance of (3.08+/-0.07) A, with a tilting angle between the heme normal and the Fe-C vector of (37+/-7) degrees, and a bending angle between the Fe-C vector and the C-O bond of (31+/-5) degrees.

Visible light can break the Fe-CO bond in Fe(II) carbonmonoxy-myoglobin (MbCO) giving an unligated product (Mb*) that is almost stable at T < 30 K. Fe K-edge polarized X-ray absorption spectra (P-XAS) of the photoproduct (T = 20 K) of an oriented single crystal (0.2 x 0.2 x 0.3 mm) of sperm whale MbCO (space group P2(1)) have been collected. By rotating the crystal the X-ray photon polarization vector has been oriented almost parallel (with an angle (alpha = 23 degrees) or perpendicular (alpha = 86 degrees) to the heme normal of each myoglobin molecule. The crystal was continuously illuminated by a white-light source during the data collection. The polarized data give novel information on the Fe-heme electronic/structural rearrangement following photolysis. The XANES (X-ray absorption near-edge structure) spectrum polarized in the direction close to the Fe-CO bond changes dramatically after photolysis, exhibiting a shift of similar to 2 eV, due to electronic relaxation of empty states of p(z) symmetry, while more subtle changes are observed in the spectrum polarized along the heme plane, sensitive to the heme-plane geometry. Changes in the pre-edge region can be interpreted to provide insight into the electronic structure of the highest occupied and lowest unoccupied molecular orbitals (HOMO-LUMO) in the MbCO --> Mb* photochemical reaction at low temperature.