The effect of pH and nitrite on the haem pocket of GLB-33, a globin-coupled neuronal transmembrane receptor of Caenorhabditis elegans.

Out of the 34 globins in Caenorhabditis elegans, GLB-33 is a putative globin-coupled transmembrane receptor with a yet unknown function. The globin domain (GD) contains a particularly hydrophobic haem pocket, that rapidly oxidizes to a low-spin hydroxide-ligated haem state at physiological pH. Moreover, the GD has one of the fastest nitrite reductase activity ever reported for globins. Here, we use a combination of electronic circular dichroism, resonance Raman and electron paramagnetic resonance (EPR) spectroscopy with mass spectrometry to study the pH dependence of the ferric form of the recombinantly over-expressed GD in the presence and absence of nitrite. The competitive binding of nitrite and hydroxide is examined as well as nitrite-induced haem modifications at acidic pH. Comparison of the spectroscopic results with data from other haem proteins allows to deduce the important effect of Arg at position E10 in stabilization of exogenous ligands. Furthermore, continuous-wave and pulsed EPR indicate that ligation of nitrite occurs in a nitrito mode at pH 5.0 and above. At pH 4.0, an additional formation of a nitro-bound haem form is observed along with fast formation of a nitri-globin.

Interaction of nitrite with ferric protoglobin from Methanosarcina acetivorans - an interesting model for spectroscopic studies of the haem-ligand interaction.

Protoglobin from Methanosarcina acetivorans (MaPgb) is a dimeric globin belonging to the same lineage of the globin superfamily as globin-coupled sensors. A putative role in the scavenging of reactive nitrogen and oxygen species has been suggested as a possible adaptation mechanism of the host organism to different gaseous environments in the course of evolution. A combination of optical absorption, electronic circular dichroism (ECD), resonance Raman (rRaman), and electron paramagnetic resonance (EPR) reveal the unusual in vitro reaction of ferric MaPgb with nitrite. In contrast to other globins, a large excess of nitrite did not induce the formation of a nitriglobin form in MaPgb. Surprisingly, the addition of nitrite in mildly acidic pH led to the formation of a stable nitric-oxide ligated ferric form of the protein (MaPgb-NO). Furthermore, the 300-700 nm ECD spectrum of ferric MaPgb is for the first time reported and discussed, showing strong differences in the Soret and Q ellipticity compared to ferric myoglobin, in line with the unusually strongly ruffled haem group of MaPgb and the related quantum-mechanical admixture of the S = 5/2 and S = 3/2 state of its ferric form. The Soret and Q ellipticity change strongly upon formation of MaPgb-NO, revealing a significant effect of the nitric-oxide ligation on the haem group and pocket. The related changes in the asymmetric pyrrole half-ring stretching vibration modes observed in the rRaman spectra give experimental support to earlier theoretical models, in which an important role of the in-plane breathing modes of the haem was predicted for the stabilization of the binding of diatomic gases to MaPgb.

GLB-3: A resilient, cysteine-rich, membrane-tethered globin expressed in the reproductive and nervous system of Caenorhabditis elegans.

The popular genetic model organism Caenorhabditis elegans (C. elegans) encodes 34 globins, whereby the few that are well-characterized show divergent properties besides the typical oxygen carrier function. Here, we present a biophysical characterization and expression analysis of C. elegans globin-3 (GLB-3). GLB-3 is predicted to exist in two isoforms and is expressed in the reproductive and nervous system. Knockout of this globin causes a 99% reduction in fertility and reduced motility. Spectroscopic analysis reveals that GLB-3 exists as a bis-histidyl-ligated low-spin form in both the ferrous and ferric heme form. A function in binding of diatomic gases is excluded on the basis of the slow CO-binding kinetics. Unlike other globins, GLB-3 is also not capable of reacting with H2O2, H2S, and nitrite. Intriguingly, not only does GLB-3 contain a high number of cysteine residues, it is also highly stable under harsh conditions (pH = 2 and high concentrations of H2O2). The resilience diminishes when the N- and C-terminal extensions are removed. Redox potentiometric measurements reveal a slightly positive redox potential (+8 ± 19 mV vs. SHE), suggesting that the heme iron may be able to oxidize cysteines. Electron paramagnetic resonance shows that formation of an intramolecular disulphide bridge, involving Cys70, affects the heme-pocket region. The results suggest an involvement of the globin in (cysteine) redox chemistry.

In Silico Identification and Experimental Validation of Novel Anti-Alzheimer's Multitargeted Ligands from a Marine Source Featuring a "2-Aminoimidazole plus Aromatic Group" Scaffold.

Multitargeting or polypharmacological approaches, looking for single chemical entities retaining the ability to bind two or more molecular targets, are a potentially powerful strategy to fight complex, multifactorial pathologies. Unfortunately, the search for multiligand agents is challenging because only a small subset of molecules contained in molecular databases are bioactive and even fewer are active on a preselected set of multiple targets. However, collections of natural compounds feature a significantly higher fraction of bioactive molecules than synthetic ones. In this view, we searched our library of 1175 natural compounds from marine sources for molecules including a 2-aminoimidazole+aromatic group motif, found in known compounds active on single relevant targets for Alzheimer's disease (AD). This identified two molecules, a pseudozoanthoxanthin (1) and a bromo-pyrrole alkaloid (2), which were predicted by a computational approach to possess interesting multitarget profiles on AD target proteins. Biochemical assays experimentally confirmed their biological activities. The two compounds inhibit acetylcholinesterase, butyrylcholinesterase, and ß-secretase enzymes in high- to sub-micromolar range. They are also able to prevent and revert ß-amyloid (Aß) aggregation of both Aß1-40 and Aß1-42 peptides, with 1 being more active than 2. Preliminary in vivo studies suggest that compound 1 is able to restore cholinergic cortico-hippocampal functional connectivity.

Design, Synthesis, and Evaluation of Donepezil-Like Compounds as AChE and BACE-1 Inhibitors.

An ecofriendly synthetic pathway for the synthesis of donepezil precursors is described. Alternative energy sources were used for the total synthesis in order to improve yields, regioselectively, and rate of each synthetic step and to reduce the coproduction of waste at the same time. For all products, characterized by an improved structural rigidity respect to donepezil, the inhibitor activity on AChE, the selectivity vs BuChE, the side-activity on BACE-1, and the effect on SHSY-5Y neuroblastoma cells viability were tested. Two potential new lead compounds for a dual therapeutic strategy against Alzheimer's disease were envisaged.

Nondenaturing polyacrylamide gel electrophoresis to study the dissociation of the p53·MDM2/X complex by potentially anticancer compounds.

A new analytical method to study the dissociation of the complexes between the oncosuppressor p53 and its negative modulators murine double-minute protein 2 (MDM2) or MDMX, is proposed. This technique is reliable to determine the dissociative power exerted by small molecules on the complex taking advantage of the appearance of migrating MDM2 or MDMX in a native polyacrylamide gel, when inhibitors are added to the complex mixture. Therefore, we propose this new approach to easily screen library of compounds, with potential pharmacological anticancer activity.

Native PAGE to study the interaction between the oncosuppressor p53 and its protein ligands.

In the present study, we investigated a new approach for studying the interaction between p53 and MDM2/X (where MDM is murine double minute protein). The method is based on the different mobility between the interacting domains of the oncosuppressor p53 and its protein ligands MDM2/X on polyacrylamide gels under native conditions. While the two proteins MDM2/X alone were able to enter the gel, the formation of a binary complex between p53 and MDM2/X prevented the gel entry. The novel technique is reliable for determining the different affinity elicited by MDM2 or MDMX toward p53, and can be useful for analyzing the dissociation power exerted by other molecules on the p53-MDM2/X complex.